Contents

Agilent AssapMAP Protein Sample Prep Workbench Handler User Guide PDF

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1 of 695

Summary of Content for Agilent AssapMAP Protein Sample Prep Workbench Handler User Guide PDF

AssayMAP Protein Sample Prep Workbench

User Guide

Original Instructions

Notices Manual Part Number D0021982 Revision A

October 2022

Copyright Agilent Technologies, Inc. 2022

No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Contact Information Agilent Technologies Inc. Automation Solutions 5301 Stevens Creek Blvd. Santa Clara, CA 95051 USA

Web: https://www.agilent.com

Contact page: https://www.agilent.com/en/contact- us/page

Documentation feedback: documentation.automation@agilent.co m

Acknowledgements Microsoft and Windows are either registered trademarks or trademarks of the Microsoft Corporation in the United States and other countries.

Warranty The material contained in this document is provided as is, and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all war- ranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information con- tained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agree- ment shall control.

Technology Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license.

Restricted Rights Legend If software is for use in the performance of a U.S. Government prime contract or subcontract, Software is delivered and licensed as Commercial computer software as defined in DFAR 252.227-7014 (June 1995), or as a commercial item as defined in FAR 2.101(a) or as Restricted computer software as defined in FAR 52.227-19 (June 1987) or any equivalent agency regulation or contract clause. Use, duplication or disclosure of Software is subject to Agilent Technologies standard commercial license terms, and non-DOD Departments and Agencies of the U.S. Government will receive no greater than Restricted Rights as defined in FAR 52.227- 19(c)(1-2) (June 1987). U.S. Government users will receive no greater than Limited Rights as defined in FAR 52.227-14 (June1987) or DFAR 252.227-7015 (b)(2) (November 1995), as applicable in any technical data.

Safety Notices

A WARNING notice denotes a haz- ard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indi- cated conditions are fully understood and met.

A CAUTION notice denotes a haz- ard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not pro- ceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

WARNING

CAUTION

iiiAssayMAP Protein Sample Prep Workbench User Guide

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii About this guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii Reporting problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

1. Getting started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Starting the workbench and logging in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Changing your password. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 About the Protein Sample Prep Workbench. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 About the Workflow Library. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 About the App Library. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 About the Utility Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 About the Literature Library. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Compliance-enabling features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Overview of software architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Exporting and importing AssayMAP methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2. Using Experiment IDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Experiment IDs and database overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Creating and managing experiment IDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Selecting an experiment ID and adding notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Generating an experiment ID report. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Closing and archiving an experiment ID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Exporting and importing experiment IDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3. Affinity Purification v3.0 User Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Preparing the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

4. Affinity Purification: Aspiration Mode v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Preparing the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

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iv AssayMAP Protein Sample Prep Workbench User Guide

5. Fractionation v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Preparing the samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

6. IMAC Cartridge Customization v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Planning the cartridge layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

7. Immobilization v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Preparing the samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

8. In-Solution Digestion: Multi-Plate v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Preparing the samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Running the Reagent Plate Setup protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Running the Digestion protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

9. In-Solution Digestion: Single Plate v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Preparing the samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

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vAssayMAP Protein Sample Prep Workbench User Guide

10. On-Cartridge Reaction v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Preparing cartridges with immobilized substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

11. Peptide Cleanup v4.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Preparing the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

12. Peptide Cleanup: Aspiration Mode v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 Preparing the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423

13. Phosphopeptide Enrichment v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 Preparing the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

14. Protein Cleanup v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 App description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468 Before you start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468 Preparing the solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472 Preparing the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479 Assay development guidelines and protocol notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488 Reference library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504

Contents

vi AssayMAP Protein Sample Prep Workbench User Guide

15. Utilities User Guides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505 Cartridge Transfer v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 Pipette Tip Transfer v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512 Reagent Aliquot v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518 Reagent Transfer v3.0 User Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 Single Liquid Addition v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542 Syringe Test v2.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549 Syringe Wash v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 System Startup/Shutdown v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574

16. Normalization v3.0 User Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585 Utility description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586 Setting up a Normalization method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590 Preparing the diluent and samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610 Assay development guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615

17. Reformatting v3.0 User Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623 Utility description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 Setting up a Reformatting method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643 Assay development guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649

18. Serial Dilution v3.0 User Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653 Utility description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654 Before you start. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654 Setting up a Serial Dilution method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658 Preparing the diluent and samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672 Running the protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674 Assay development guidelines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679

19. Emergency stops and pauses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683

vii

Preface This preface contains the following topics: About this guide on page viii Reporting problems on page x

Preface About this guide

viii AssayMAP Protein Sample Prep Workbench User Guide

About this guide

Overview

This guide includes the user guides for the applications and utilities in the AssayMAP Protein Sample Prep Workbench.

Software version

This guide documents the following software versions: Protein Sample Prep Workbench 4.0 VWorks Automation Control 14.1.1

Accessing the user guides

You can find the user guides for the AssayMAP applications and utilities in the Protein Sample Prep Workbench.

The Literature Library provides links to the user guides.

ix

Preface About this guide

AssayMAP Protein Sample Prep Workbench User Guide

The app quick start guides are available in the App Library and additional utility guides are available in the Utilities Library.

Preface Reporting problems

x AssayMAP Protein Sample Prep Workbench User Guide

Reporting problems

If you find a problem with the AssayMAP Bravo Platform, contact Agilent Automation Solutions Technical Support. For contact information, go to https://www.agilent.com/ en/contact-us/page.

For instructions on how to resolve common error messages, see the AssayMAP Bravo Platform Error Recovery Guide.

To report problems with Have the following information ready

Hardware Instrument serial number from the Bravo serial number label

Short description of the problem

Software Instrument serial number from the Bravo serial number label

Short description of the problem Relevant software version number (for example,

automation control software, diagnostics software, and firmware)

Error message text (or screen capture of the error message dialog box)

Relevant files, such as log files

AssayMAP Cartridges Cartridge type Lot number Short description of the problem

1

1 Getting started This section contains the following topics: Starting the workbench and logging in on page 2 Changing your password on page 3 About the Protein Sample Prep Workbench on page 4 About the Workflow Library on page 5 About the App Library on page 7 About the Utility Library on page 9 About the Literature Library on page 10 Compliance-enabling features on page 13 Overview of software architecture on page 15 Exporting and importing AssayMAP methods on page 17

1 Getting started Starting the workbench and logging in

2 AssayMAP Protein Sample Prep Workbench User Guide

Starting the workbench and logging in

For instructions on how to start up the AssayMAP Bravo Platform, see System Startup/Shutdown v3.0 User Guide on page 574.

Starting the Protein Sample Prep Workbench

VWorks administrator, technician, operator, or guest privileges are required to log in to the software. Contact your administrator if you need login credentials.

To start the Protein Sample Prep Workbench:

1 Double-click on the Windows desktop. The Protein Sample Prep Workbench window opens.

2 Click one of the following buttons in the Workbench window: Workflow Library Application Library Utility Library Literature Library The VWorks software starts and the VWorks Login window opens.

3

1 Getting started Changing your password

AssayMAP Protein Sample Prep Workbench User Guide

3 In the VWorks Login window, type your Login and Password, and then click OK. The VWorks window opens. After a few seconds, the selected Library page opens in the VWorks window. For a description of each Library, see About the Protein Sample Prep Workbench on page 4.

Changing your password

The following procedure is applicable if Open Lab Control Panel is configured to use Internal authentication. If Open Lab Control Panel is configured to use Windows Domain, see the Windows documentation for instructions on how to change your password. If you need help changing your password, contact your administrator.

Changing your password in the OpenLab Control Panel

To log in to Control Panel and change your VWorks user password:

1 Double-click on the Windows desktop to start OpenLab Control Panel.

Alternatively, select Start ( ) > All Apps > Agilent Technologies > Control Panel.

2 In the Control Panel login window, enter your user name and password, and click Log In.

1 Getting started About the Protein Sample Prep Workbench

4 AssayMAP Protein Sample Prep Workbench User Guide

If you log in as a VWorks technician, operator, or guest, the Control Panel opens to the My Settings page.

3 In the ribbon at the top of the My Settings page, click Change My Password. 4 In the Change My Password dialog box, type the Old password, type the new

password in the New password and Confirm new password boxes, and then click OK.

5 If you have finished viewing the My Settings page, close the Control Panel.

About the Protein Sample Prep Workbench

The Protein Sample Prep Workbench includes the following: Workflow Library. For details, see About the Workflow Library on page 5. Application Library. For details, see About the App Library on page 7. Utility Library. For details, see About the Utility Library on page 9. Literature Library. For details, see About the Literature Library on page 10.

5

1 Getting started About the Workflow Library

AssayMAP Protein Sample Prep Workbench User Guide

Major and minor revisions of apps and utilities

The version of the Protein Sample Prep Workbench, each application, and each utility is designated by a decimal number, where Major revision or original release is designated by the number before the decimal

point. A major revision is a change to the protocol and may also include changes to the interface (AssayMAP form).

Minor revision is designated by the number after the decimal point. Minor revisions indicate a change only to the AssayMAP form and does not impact how the protocol runs.

For example, version 1.0 designates an original release of the application or utility, version 1.1 designates a change to the form only, version 2.0 designates a change to the protocol, and so forth. A major version automatically resets the minor number to 0.

About the Workflow Library

The Workflow Library is a collection of protein sample preparation workflows. Each workflow consists of a set of applications that are performed in sequence to complete an experiment. In addition, the workbench contains links to utilities that facilitate sample and reagent preparation, the transition between applications, and the transition between the output of the final application and the input of the analytical instrument.

1 Getting started About the Workflow Library

6 AssayMAP Protein Sample Prep Workbench User Guide

General Workflows

Post-Translational Modification Workflows

Workflow Enables the

Affinity Purification Creation of custom affinity cartridges that can be used to enrich target molecules. The workflow consists of the following applications: Immobilization. For details, see the Immobilization v3.0 User Guide on

page 193. Affinity Purification. For details, see the Affinity Purification v3.0 User Guide

on page 45.

Peptide Sample Prep Digestion of proteins, the desalting of the peptide resulting from the digestion, and optionally the fractionation of the peptide mixture. The workflow consists of the following applications: In-Solution Digestion. For details, see the In-Solution Digestion: Single Plate

v2.0 User Guide on page 275. Peptide Cleanup. For details, see the Peptide Cleanup v4.0 User Guide on

page 355. Fractionation. For details, see the Fractionation v2.0 User Guide on page 125.

Rapid Antibody Digestion

Desalting of denatured antibodies, and the rapid digestion of these antibodies. The workflow consists of the following applications: Affinity Purification. For details, see the Affinity Purification v3.0 User Guide

on page 45. In-Solution Digestion. For details, see theIn-Solution Digestion: Single Plate

v2.0 User Guide on page 275. Protein Cleanup. For details, see the Protein Cleanup v3.0 User Guide on

page 467.

Workflow Enables the

Phosphopeptide Enrichment

Digestion of proteins, the desalting of the peptide resulting from the digestion, and the enrichment of the phosphopeptides. The workflow consists of the following applications: In-Solution Digestion. For details, see the In-Solution Digestion. For details, see

the In-Solution Digestion: Single Plate v2.0 User Guide on page 275. Peptide Cleanup. For details, see the Peptide Cleanup v4.0 User Guide on

page 355. Phosphopeptide Enrichment. For details, see the Phosphopeptide Enrichment

v3.0 User Guide on page 425.

7

1 Getting started About the App Library

AssayMAP Protein Sample Prep Workbench User Guide

Evosep Workflow

About the App Library

The App Library contains a collection of ready-to-use sample processing applications that can be used individually or in various combinations to address your experimental needs.

Available applications

Workflow Enables the

Evosep 96AM Workflow

Automation of the pipetting steps for the Evotip Pure loading protocol. For details, see the Evosep - 96AM Workflow Quick Start Guide at Quick start guides on page 1.

Application Automates the See

Affinity Purification v3.0

Affinity purification of target molecules using AssayMAP cartridges. This is the recommended Affinity Purification application.

Affinity Purification v3.0 User Guide on page 45

1 Getting started About the App Library

8 AssayMAP Protein Sample Prep Workbench User Guide

Affinity Purification: Aspiration Mode v3.0

Affinity purification of target molecules using AssayMAP cartridges. The sample load and wash steps in this protocol are drawn into rather than pushed through the cartridges, which is in contrast to the Affinity Purification v3.0 protocol. This application may be useful for certain workflows, but is more prone to clogging.

Affinity Purification: Aspiration Mode v3.0 User Guide on page 89

Fractionation v2.0 Stepwise fractionation of samples from AssayMAP cartridges in up to six fractions using buffers with increasing ionic strength, increasing pH, or increasing organic concentration.

Fractionation v2.0 User Guide on page 125

IMAC Cartridge Customization v2.0

Stripping and charging of AssayMAP Fe(III)-NTA cartridges with the desired metal of choice for immobilized metal affinity chromatography (IMAC) experiments.

IMAC Cartridge Customization v2.0 User Guide on page 159

Immobilization v3.0 Immobilization of antibodies and other ligands to PAW, PGW, or SAW cartridges.

Immobilization v3.0 User Guide on page 193

In-Solution Digestion: Single Plate v2.0

Digestion of 8 to 96 protein samples in a 96-well microplate. The protocol includes five generic liquid-handling steps that successively transfer a reagent from a reagent plate at deck location 5, 6, 7, 8, or 9, into the Sample Plate at deck location 4.

In-Solution Digestion: Single Plate v2.0 User Guide on page 275

In-Solution Digestion: Multi- Plate v2.0

Digestion of 8 to 384 protein samples in up to four 96-well microplates in a single run.

In-Solution Digestion: Multi-Plate v2.0 User Guide on page 235

On-Cartridge Reaction v2.0

Reactions on AssayMAP cartridges that contain immobilized target molecules.

On-Cartridge Reaction v2.0 User Guide on page 309

Peptide Cleanup v4.0

Cleanup of peptide samples using AssayMAP cartridges. This is the recommended Peptide Cleanup application.

Peptide Cleanup v4.0 User Guide on page 355

Peptide Cleanup: Aspiration Mode v3.0

Cleanup of peptide samples using AssayMAP cartridges. The sample load and wash steps in this protocol are drawn into rather than pushed through the cartridges, which is in contrast to the Peptide Cleanup v4.0 protocol. This application may be useful for certain workflows, but is more prone to clogging.

Peptide Cleanup: Aspiration Mode v3.0 User Guide on page 393

Phosphopeptide Enrichment v3.0

Phosphopeptide enrichment using the AssayMAP Fe(III)-NTA or TiO2 cartridges.

Phosphopeptide Enrichment v3.0 User Guide on page 425

Protein Cleanup v3.0

Cleanup of protein samples using the AssayMAP RP-W cartridges.

Protein Cleanup v3.0 User Guide on page 467

Application Automates the See

9

1 Getting started About the Utility Library

AssayMAP Protein Sample Prep Workbench User Guide

About the Utility Library

The Utility Library is a collection of protocols that automate specific tasks (for example, liquid handling) to prepare the system for an application protocol, transition between applications in a workflow, or transition between an application and loading onto an analytical device. For example, the System Startup/Shutdown utility initializes the AssayMAP Bravo Platform, washes the syringes, and primes the wash lines and the wash station.

Utility Description See

Cartridge Transfer v2.0 Moves full columns of AssayMAP cartridges from the source cartridge rack to the 96AM Cartridge & Tip Seating Station.

Cartridge Transfer v2.0 User Guide on page 506

Normalization v3.0 Automates the normalization of up to 96 samples in microplate format.

Normalization v3.0 User Guide on page 585

Pipette Tip Transfer v2.0 Moves full columns of disposable pipette tips from the source tip box to the 96AM Cartridge & Tip Seating Station.

Pipette Tip Transfer v2.0 User Guide on page 512

Reagent Aliquot v2.0 Aliquots a reagent from a single bulk reagent reservoir into 112 columns of a microplate or reservoir. The utility uses a single column of eight Agilent 250-L pipette tips to prepare the aliquots.

Reagent Aliquot v2.0 User Guide on page 518

1 Getting started About the Literature Library

10 AssayMAP Protein Sample Prep Workbench User Guide

About the Literature Library

The Literature Library provides links to the user documentation for the AssayMAP Bravo Platform.

Reagent Transfer v3.0 Transfers 112 columns of reagents in parallel from a reagent source plate or reservoir into a 96-well plate.

Reagent Transfer v3.0 User Guide on page 525

Reformatting v3.0 Transfers solutions from any well in one microplate to any well in another microplate.

Reformatting v3.0 User Guide on page 623

Serial Dilution v3.0 Creates a Serial Dilution plate with up to 24 dilutions and up to 5 replicates.

Serial Dilution v3.0 User Guide on page 653

Single Liquid Addition v2.0 Adjusts the buffer composition or adds a component to up to four sample plates.

Single Liquid Addition v2.0 User Guide on page 542

Syringe Test v2.0 Verifies the integrity of the probes and syringes in the Bravo 96AM Head.

Syringe Test v2.0 User Guide on page 549

Syringe Wash v3.0 Washes the syringes after an application or utility run with the solution of your choice for the specified number of wash cycles to avoid run to run cross- contamination.

Syringe Wash v3.0 User Guide on page 567

System Startup v3.0 Initializes the AssayMAP Bravo Platform, washes the syringes, and primes the wash station and wash lines.

System Startup/Shutdown v3.0 User Guide on page 574

System Shutdown v3.0 Prepares the AssayMAP Bravo Platform for idle time by washing the syringes and then aspirating 200 L of Syringe Storage Liquid into the syringes.

System Startup/Shutdown v3.0 User Guide on page 574

Utility Description See

11

1 Getting started About the Literature Library

AssayMAP Protein Sample Prep Workbench User Guide

General Help section of Literature Library

Title Description

Using the Protein Sample Prep Workbench

Provides an overview of the workbench features.

AssayMAP Bravo Platform Getting Started Guide

Provides an overview of the platform and a detailed description of the AssayMAP liquid-handling head.

Bravo Platform User Guide Explains how to set up and operate the Bravo Platform and how to configure accessories.

Labware Reference Guide Provides detailed information and a photo of each labware option for the workbench applications.

Syringe Replacement Guide Provides the procedures for replacing damaged syringes in the AssayMAP head.

Error Recovery Guide Provides guidelines for how to recover from the more common error messages that may occur when using the AssayMAP Bravo Platform.

Wash Station Maintenance Guide

Provides the procedures for inspecting the chimneys and replacing damaged chimneys.

AssayMAP Bravo Platform Installation Guide

Explains how to install the AssayMAP Bravo Platform, including configuring the deck accessories and setting the teachpoints.

1 Getting started About the Literature Library

12 AssayMAP Protein Sample Prep Workbench User Guide

App & Utility Guides section of Literature Library

The Protein Sample Prep Workbench user guides describe how to prepare and run protocols for various sample prep applications and utilities. The topics are for both routine operators and assay developers.

VWorks Knowledge Base Provides the help system for the VWorks software, including: Configuration and administration guides and quick references for VWorks

Networked, VWorks Plus, and VWorks Standard editions VWorks Setup Guide VWorks User Guide Bravo safety, installation, user, and quick-start guides User guides for other VWorks-controlled devices

Title Description

13

1 Getting started Compliance-enabling features

AssayMAP Protein Sample Prep Workbench User Guide

Compliance-enabling features

The combination of features in VWorks Plus 14.1.1.1 and Protein Sample Prep Workbench 4.0 help enable compliance with Part 11 of Title 21 of the Code of Federal Regulations (21 CFR Part 11). The FDA rules and guidelines for compliant electronic records and computerized systems require secure data handling, including: Data security. Physical protection of data by limiting access to the system and

preventing unauthorized access. Data integrity. Protecting raw data and metadata and preventing these from

unauthorized modification, and linking raw data and results to reproduce the original results at any time, for example, in an audit situation, and document each new result copy.

Audit traceability. Documenting who did what to the results and when.

VWorks Plus features that support compliance

VWorks Plus compliance-enabling features include user authentication, content management, tamper detection, audit trails, electronic signatures, and activity logs. For a description of these features, see the Using compliance features chapter in the VWorks Automation Control Setup Guide.

Workbench features that support compliance

The Protein Sample Prep Workbench uses experiment IDs and methods to support traceability and data integrity. An experiment ID and a method are required to run any application or utility in the compliance-enabled Protein Sample Prep Workbench. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. Electronic signatures are required to close an experiment ID. The software automatically generates a report when an experiment ID is closed. A report describes who did what and when for a given experiment ID. For details, see Using Experiment IDs on page 23.

A method is a comprehensive collection of saved settings for an application or utility that you can use to run the application or utility. Methods in the OpenLab Content Management storage can be used to run an app or utility protocol. Any changes to an existing method must be saved using a new name before the revised method can be used to run an app or utility protocol. For detailed procedures, see the user guide for the app or utility.

Roles and privileges

The VWorks administrator, technician, and operator roles have the following privileges in the compliance-enabled Workbench.

1 Getting started Compliance-enabling features

14 AssayMAP Protein Sample Prep Workbench User Guide

Figure VWorks Technician privileges for creating and running methods

VWorks role Privileges for experiment IDs and methods

VWorks operator Experiment IDs Select an experiment ID to run an app or utility Add notes to an experiment ID Generate an experiment ID report Export experiment IDs Methods Select a method to run an app or utility Export a method

VWorks technician All the privileges of a VWorks operator, plus the following: Experiment IDs Create experiment IDs and edit the description of experiment IDs that have the

Not Yet Used status Close experiment IDs and post a signature when closing Import experiment IDs Methods Create and save methods Import methods

IMPORTANT Any new or modified method must be saved using a unique name before it can be run, as the following figure shows.

VWorks administrator

All the privileges of a VWorks operator and technician, plus the following: Archive closed experiment IDs Delete experiment IDs that have a Not Yet Used status Log in to Content Browser and edit the project, including deleting files

15

1 Getting started Overview of software architecture

AssayMAP Protein Sample Prep Workbench User Guide

Overview of software architecture

The AssayMAP Bravo Platform requires the following software, which runs on a single computer workstation: Agilent Protein Sample Prep Workbench. A collection of simple form-based user

interfaces for running the applications and utilities that control the AssayMAP Bravo Platform. Each application and utility has default methods that can be altered and saved. The Protein Sample Prep Workbench runs within the VWorks software.

Microsoft Excel. Required for the Workbench method setup tools, reagent volume calculators, and Syringe Test data analysis tool.

Agilent VWorks software 14.1.1. A combination of Agilent OpenLab software components and Agilent VWorks software that provides the user management and instrument control for the AssayMAP Bravo Platform. The software is available in the following editions.

The following figures show the software architecture for each VWorks edition. As the figures show, the OpenLab components include a Control Panel, which is the user interface for the Shared Services. The Shared Services, such as user access, software licenses, and storage are configured initially when the software is installed. For more details on these OpenLab components, see the VWorks Plus Configuration and Administration Guide or VWorks Standard Configuration and Administration Guide.

VWorks edition File storage type Compliance enabled?

VWorks Plus Content Management Note: A system or VWorks administrator can use the Content Browser to view and edit the project structure and contents at /VWorks Projects/VWorks/

Yes

VWorks Standard Local file system Note: Any user can access the project files directly using the Windows File Explorer at C:\OLSS Projects\VWorks Projects\VWorks\

No

1 Getting started Overview of software architecture

16 AssayMAP Protein Sample Prep Workbench User Guide

Figure Architecture of VWorks Plus and Protein Sample Prep Workbench

Figure Architecture of VWorks Standard and Protein Sample Prep Workbench

The VWorks files, such as device files, profiles, labware, protocols, and the like are stored as .roiZip archives. All file modifications must be handled within the VWorks software.

IMPORTANT The VWorks software cannot reload files (.roiZip extension) that have been modified or renamed outside of the VWorks software.

17

1 Getting started Exporting and importing AssayMAP methods

AssayMAP Protein Sample Prep Workbench User Guide

Exporting and importing AssayMAP methods

A method is a comprehensive collection of saved settings that you can use to run a Workbench application or utility. Each application and utility in Protein Sample Prep Workbench 4.0 has default methods that you can use. Each app and utility user guide describes how to select and how to create methods. The VWorks export and import features enable the sharing of customized AssayMAP methods with users on a different computer. This topic describes how to export and import AssayMAP methods, one method at a time.

Prerequisites

Both computers must be running the following software: VWorks software 14.1.1 Protein Sample Prep Workbench 4.0 VWorks technician privileges or greater are required to import AssayMAP method files into the Shared Services storage. No overwrite is allowed for AssayMAP method files that already exist in Shared Services storage. The VWorks menu bar must be visible to access the export and import commands.

Displaying the VWorks Tool menu

To display the VWorks main window, do one of the following: If the Protein Sample Prep Workbench is not currently running, start the VWorks

software and log in when prompted to do so.

If the menubar is not visible in the VWorks main window, right-click the window and select Menubar in the shortcut menu that appears.

If the Protein Sample Prep Workbench is already running, use the following procedure to turn off Full Screen mode so that the VWorks File menu is visible.

To turn off Full Screen mode:

1 In any app or utility, click in the navigation pane of the form.

1 Getting started Exporting and importing AssayMAP methods

18 AssayMAP Protein Sample Prep Workbench User Guide

2 Ensure that the VWorks menu bar is visible above the Workbench form, as the following figure shows.

Figure Example of VWorks Menubar visible in Full Screen off mode

AssayMAP method file storage

The AssayMAP method files are stored in the following Shared Services locations.

The AM Methods folder stores the method files (.mth) for each app and utility in a corresponding subfolder of the same name. The file names for the default settings are the same for multiple apps and utilities. However, only the app or utility used to create a method can open that method. For example, AM Affinity Purification 3.0 subfolder contains the Affinity Purification methods:

25uL Cartridge Default Settings.mth 5uL Cartridge Default Settings.mth

AM Peptide Cleanup 4.0 subfolder contains the Peptide Cleanup methods: 25uL Cartridge Default Settings.mth 5uL Cartridge Default Settings.mth

AM Reagent Transfer v3.0 subfolder contains the Reagent Transfer method: Default Settings.mth

VWorks edition Storage location

VWorks Plus /VWorks Projects/VWorks/AM Methods/

VWorks Standard C:/OLSS Projects/VWorks Projects/VWorks/AM Methods/

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1 Getting started Exporting and importing AssayMAP methods

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AM Syringe Wash v3.0 subfolder contains the Syringe Wash method: Default Settings.mth

To help prevent accidentally importing a method file into the wrong subfolder, the software prepends the corresponding app or utility folder name to the file name when you export a method.

IMPORTANT Agilent recommends that you retain the exported file name assigned by the software until that file is imported to the correct subfolder. If desired, you can remove the folder name from a file name when you import it, after navigating to the correct subfolder with the same folder name.

Exporting an AssayMAP method

To export an AssayMAP method file (.mth): 1 In the VWorks window, click File > Export Misc File. The Open File dialog box

appears.

2 In the Open File dialog box: a Click AssayMAP Method Files (*.mth) in the Files of type list.

b Use the button to navigate to the AM Methods app or utility subfolder. c Select the method file to be exported, and then click Open.

The Export Miscellaneous File To dialog box opens.

1 Getting started Exporting and importing AssayMAP methods

20 AssayMAP Protein Sample Prep Workbench User Guide

3 In the Export Miscellaneous File To dialog box:

IMPORTANT Agilent recommends retaining the folder name as part of the exported file name to help ensure that the file is imported to the correct method subfolder for the corresponding app or utility.

a Notice that the proposed File name includes the name of the app or utility subfolder of the method file you are exporting.

b Ensure that the Save as type is AssayMAP Method Files (*.mth). c Navigate to the export location, and then click Save.

Importing an AssayMAP method

To import an AssayMAP method file (.mth): 1 In the VWorks window, click File > Import Misc File. The Select a Miscellaneous File

for Import dialog box opens.

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1 Getting started Exporting and importing AssayMAP methods

AssayMAP Protein Sample Prep Workbench User Guide

2 In the Select a Miscellaneous File for Import dialog box: a Ensure that the AssayMAP Method Files (*.mth) file type is selected. b Navigate to the file location, select the method file to be imported, and then

click Open. 3 In the Save File As dialog box that opens, do the following:

a Use the button to navigate to the corresponding AM Methods app or utility subfolder. Ensure that you select the subfolder for the app or utility that created this method.

b Ensure the file name is different from the other files in the folder. You can remove the subfolder name from the method file name after you have selected the correct app or utility subfolder.

c Ensure that the Files of type box specifies AssayMAP Method Files (*.mth), and then click Save. If a file of the same name already exists in this folder, a message tells you that you cannot overwrite the existing file.

1 Getting started Exporting and importing AssayMAP methods

22 AssayMAP Protein Sample Prep Workbench User Guide

4 VWorks Plus only. The Audit Comment dialog box opens. Select or type the audit comment, and then click OK. Note: Imported AssayMAP method files are exempt from changes in record state. An audit trail is always logged for exempt state records.

23

2 Using Experiment IDs This section contains the following topics: Experiment IDs and database overview on page 24 Creating and managing experiment IDs on page 26 Selecting an experiment ID and adding notes on page 30 Generating an experiment ID report on page 32 Closing and archiving an experiment ID on page 35 Exporting and importing experiment IDs on page 42

2 Using Experiment IDs Experiment IDs and database overview

24 AssayMAP Protein Sample Prep Workbench User Guide

Experiment IDs and database overview

An experiment ID is a database record that captures the steps executed and the settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. You use the Experiments Editor to create and manage experiment IDs. The following figure shows an example of how an experiment ID can be used to record the steps performed and settings used during the course of a sample prep workflow.

Figure Example of an Experiment ID workflow

Experiment IDs are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Software prerequisites

Before the Experiments Editor is available for use, a connection to the VWorks Experiments database must be established. For instructions, see Setting up and connecting VWorks Experiments database on page 101 in the VWorks Automation Control Setup Guide.

Workflow overview for experiment IDs

Step For this task... See...

1 Use Experiments Editor to create an experiment ID. Note: You can open the Experiments Editor from any Workbench app or utility.

Creating and managing experiment IDs on page 26

2 In the app or utility, select the experiment ID and method, and then start the run. During the protocol run, the software updates the database with the specified information for the selected experiment ID.

User guide for the workbench app or utility

25

2 Using Experiment IDs Experiment IDs and database overview

AssayMAP Protein Sample Prep Workbench User Guide

3 If applicable, add notes while the experiment ID has an Open status. The preceding figure shows an example of a note for a denaturation step.

Adding notes to an experiment ID on page 30

4 Generate an experiment ID report. Generating an experiment ID report on page 32

5 Close the experiment ID. An experiment ID report is automatically generated.

Closing an experiment ID on page 35

6 Archive closed experiments to help prevent the Experiments database from being overloaded.

Closing and archiving an experiment ID on page 35

7 Periodically make a backup copy of the Experiments database and store the backup in a secure location. Ensure that the backup copy includes: Exported data currently in the

Experiments database Archived experiment ID data.

Exporting and importing experiment IDs on page 42

Step For this task... See...

2 Using Experiment IDs Creating and managing experiment IDs

26 AssayMAP Protein Sample Prep Workbench User Guide

Creating and managing experiment IDs

You must have VWorks administrator or technician privileges to create experiments.

Creating an experiment ID

To create an experiment ID: 1 In the navigation pane of an app or utility, click Experiments Editor. The

Experiments Editor window opens.

2 In the Experiments Editor window, click Create.

3 In the Create New Experiment dialog box that opens: a Type a name in the Experiment ID box. b In the Experiment Description box, type a description for this experiment ID. c Click OK. Note: The software assigns each experiment ID a hidden unique identifier (GUID).

4 In the Experiments Editor window, notice the following: The new experiment ID appears in the Experiment ID table.

27

2 Using Experiment IDs Creating and managing experiment IDs

AssayMAP Protein Sample Prep Workbench User Guide

The Status column displays Not yet used for any experiment IDs that have not been associated with

any protocol runs. Open if the experiment ID has had data added to it from at least one

application run. Closed for any closed experiment IDs only if the Show closed

experiments check box is selected. The Experiment Description box displays the comments for the selected

experiment ID.

Editing descriptions for an experiment ID

You may edit the descriptions of experiment IDs that have the Not Yet Used or Open status. Closed experiment IDs cannot be edited. VWorks technician or administrator privileges are required for this procedure. VWorks Plus only. You may edit the description of an experiment ID only if it has the Not Yet Used status.

To edit the description for an experiment ID: 1 In the Experiments Editor window, select the Experiment ID, and then click Edit

description.

2 Using Experiment IDs Creating and managing experiment IDs

28 AssayMAP Protein Sample Prep Workbench User Guide

2 In the Edit Description dialog box, type or modify the description, and then click OK.

In the Experiments Editor window, select the experiment ID to view the corresponding description in the Experiment Description box.

Deleting an experiment ID

An administrator may delete an experiment ID if it has a Not Yet Used or Open status. Closed experiments cannot be deleted. VWorks Plus. Only an experiment ID with a Not Yet Used status can be deleted.

To delete an experiment ID: 1 In the Experiment Editor window, select the Experiment ID, and then click Delete.

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2 Using Experiment IDs Creating and managing experiment IDs

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2 In the confirmation message that appears, click Yes to delete this experiment ID. The experiment ID no longer appears in the Experiment ID table.

2 Using Experiment IDs Selecting an experiment ID and adding notes

30 AssayMAP Protein Sample Prep Workbench User Guide

Selecting an experiment ID and adding notes

Selecting an experiment ID for a run

To select an experiment ID for a run: 1 In a form, click the Select Experiment button. The Experiments Editor window

opens. 2 In the Experiments Editor window, select the Experiment ID and then click Use

Selected.

The selected experiment ID will be used for the next run initiated from the form.

Adding notes to an experiment ID

You can add notes to an open experiment ID, for example, to describe an off-deck denaturation step. The notes that you add will appear in any reports generated for the experiment ID.

To use the Add Experiment Note button: 1 Ensure that the Experiment ID is selected in form, and then click

. The Add Note dialog box opens.

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2 Using Experiment IDs Selecting an experiment ID and adding notes

AssayMAP Protein Sample Prep Workbench User Guide

2 In the Note area, type the note, and then click OK.

To select an experiment ID and add a note: 1 Do one of the following to open the Experiments Editor:

In an app or utility form, click Select Experiment. In the VWorks window, click Tools > Experiments Editor.

2 In the Experiments Editor window, select the Experiment ID, and then click Add Note.

The Add Note dialog box opens.

3 In the Note area, type the note, and then click OK.

2 Using Experiment IDs Generating an experiment ID report

32 AssayMAP Protein Sample Prep Workbench User Guide

Generating an experiment ID report

The software automatically generates an experiment ID report when the experiment ID is closed. The following procedure describes how to generate a report for any experiment ID selected in the Experiments Editor.

Creating a report for a selected experiment ID

To create an experiment ID report: 1 Open the Experiments Editor using one of the following methods:

In a VWorks form, click the Experiments Editor or Select Experiment button. In the VWorks window, click Tools > Experiments Editor.

2 Optional. To include closed experiment IDs in the Experiments Editor window, select the Show closed experiments check box.

3 Select the Experiment ID and then click Create Report.

4 In the Save As dialog box, type a file name for the report, select a storage location, and then click Save. The software saves the report as a pdf file.

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2 Using Experiment IDs Generating an experiment ID report

AssayMAP Protein Sample Prep Workbench User Guide

Note: The VWorks software uses the Microsoft Print to PDF virtual printer to generate .pdf files. This virtual printer is installed on all Windows 10 systems by default. If the virtual print has been removed, the report generation feature will not work.

Experiment ID report contents

The following figure shows an example of an experiment ID report, the following table describes the report contents.

Figure Example experiment ID report

Table Report contents

2 Using Experiment IDs Generating an experiment ID report

34 AssayMAP Protein Sample Prep Workbench User Guide

Item Description

Report header Date that the report was generated Experiment ID, creation date, and user log in of experiment ID

creator Description provided for the experiment

Archival status and history, if applicable

Archival status of experiment ID: Date of archival or restoration User who archived or restored File name and path of the archived to or restored from location

Application and utility details Application or utility name Iteration of the protocol run, and if it was simulated.

Each iteration is listed separately, and ordered by launch time. Individual steps within an application or utility iteration are separated by a single blank line.

Log in of the user Date and time run was started Method name Number of full columns of cartridges Labware selection for each deck location Step details for each step in the application

Notes Date and time the note was appended and the user login Note contents

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2 Using Experiment IDs Closing and archiving an experiment ID

AssayMAP Protein Sample Prep Workbench User Guide

Closing and archiving an experiment ID

You must have VWorks administrator or technician privileges to change the status of an experiment ID from open to closed or from closed to archived. VWorks Plus. Only a VWorks administrator may archive an experiment ID.

IMPORTANT To ensure that you can recover the experiment ID data if the computer crashes or data get corrupted or lost, keep a backup copy of all data from the Experiments database and any archived experiment ID files in a secure location.

Closing an experiment ID

To close an experiment ID: 1 In the Experiment Editor, select the Experiment ID that you want to close. It must

have an Open status. Click Close Status.

Experiment ID status Description

Closed Automatically generates an experiment ID report and stores it in Shared Services storage. The experiment ID is used as the file name of the report.

Cannot be reopened. Cannot have data added and it cannot be used for any subsequent application

runs. If e-signatures are enabled, closed status is pending until all required signatures are completed. While closed status is pending, the experiment ID cannot have data added and it cannot be used for any application runs.

Archived Saves experiment ID or IDs to a file and erases associated experiment ID data from the active Experiments database. You can archive individual or multiple experiment IDs to a given archive file.

May be restored to the Experiments database as a closed experiment ID or IDs. Helps prevent the Experiments database from being overloaded.

You should periodically archive closed experiment IDs that are no longer active, and keep a backup copy in a secure location.

You may only archive closed experiment IDs. You cannot edit, delete, or export archived experiments.

2 Using Experiment IDs Closing and archiving an experiment ID

36 AssayMAP Protein Sample Prep Workbench User Guide

A message warns you that a closed experiment ID cannot be reopened. Click Yes to continue.

VWorks Plus. If e-signatures are enabled, the status changes to Signatures pending for closure since . Only after all the required signatures are completed will the status change to Closed.

2 VWorks Plus only. If e-signatures are enabled and your login credentials allow you to sign at this stage, enter the following in the E-Sign dialog box, and then click Sign: Meaning. Select the meaning from the list. Comment. Type a comment about why you are closing this experiment ID. Login and Password. Type your VWorks login credentials.

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2 Using Experiment IDs Closing and archiving an experiment ID

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A Signature(s) posted successfully message displays.

If you do not have the credentials to sign at this stage, you may want to notify the other signatories that their signatures are pending.

3 To view closed experiment IDs in the Experiments Editor, select the Show closed experiments check box at the top of the window.

The Status column displays Closed at <date and time> for the closed experiment IDs. If the experiment ID was restored from an archive, the Status column displays Closed without the date-time stamp.

Archiving an experiment ID

To archive an experiment ID: 1 In the Experiments Editor window, select the Show closed experiments check box.

2 Using Experiment IDs Closing and archiving an experiment ID

38 AssayMAP Protein Sample Prep Workbench User Guide

2 Select the Closed experiment ID or IDs to be archived, and then click Archive. Note: To select multiple items, use SHIFT+click or CTRL+click.

3 When the confirmation message opens stating that all data associated with the archived experiment ID or IDs will be saved to a file and that the data will be erased from the database, do one of the following: Click No to cancel the archive operation. Click Yes to archive the selected experiment IDs.

An Archived Selected Experiment IDs message opens and lists the storage location and file name (<datetimestamp>.expTags), as the following example shows.

Viewing the archived experiment IDs

To view the archived experiment IDs: 1 In the VWorks window, click Tools > Experiments Archive.

The Experiments Archive dialog box opens and displays all archived experiment IDs.

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2 Using Experiment IDs Closing and archiving an experiment ID

AssayMAP Protein Sample Prep Workbench User Guide

2 To filter the list of archived experiment IDs, type the filter text in the Find box, and then click Find. This filter is not case-sensitive as the following example shows.

3 Select the experiment ID to view the description in the Experiment Description box.

Creating reports of archived experiment IDs

To create a report for an archived experiment ID: 1 In the VWorks window, click Tools > Experiments Archive. 2 In the Experiments Archive dialog box, locate and select the Experiment ID, and then

click Create Report.

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40 AssayMAP Protein Sample Prep Workbench User Guide

3 In the Save As dialog box, specify the file name and storage location, and then click Save.

Restoring archived experiment IDs to the database

You must have VWorks technician or administrator to restore archived experiment IDs. When you restore an archived experiment ID to the database, the status of the experiment ID changes to closed.

To restore archived experiment IDs to the Experiments database: 1 In the VWorks window, click Tools > Experiments Archive. 2 In the Experiments Archive dialog box, locate and select the experiment ID or IDs,

and then click Restore.

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2 Using Experiment IDs Closing and archiving an experiment ID

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The Import/Restore Completed message appears and lists the restored experiment IDs.

The Experiments Editor lists the restored experiment IDs with a Closed status and no date-time stamp.

2 Using Experiment IDs Exporting and importing experiment IDs

42 AssayMAP Protein Sample Prep Workbench User Guide

Exporting and importing experiment IDs

IMPORTANT To ensure that you can recover the experiment ID data if the computer crashes or data get corrupted or lost, keep a backup copy of all data from the Experiments database and any archived experiment IDs in a secure location.

You can export active experiment IDs from the Experiments database to files in the local file system. You can export the data for individual or multiple experiment IDs to a single file. The exported data does not include archived experiment IDs. When importing any previously exported experiment IDs, the software performs tamper detection to verify that no changes were made to the exported experiment ID files. You should periodically export the data for all active experiment IDs to create a backup copy of the database. You can import the backup copy to recover the experiment ID data in case the data become damaged or lost.

Exporting experiment IDs

You can export experiment IDs that have a status of Not Yet Used, Open, or Closed.

To export experiment IDs: 1 Do one of the following to open the Experiments Editor:

In an app or utility form, click Experiments Editor or Select Experiment. In the VWorks window, click Tools > Experiments Editor.

2 In the Experiments Editor window, select the experiment IDs, and then click Export.

3 In the Save As dialog box, select the storage location, type a file name, and then click Save.

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2 Using Experiment IDs Exporting and importing experiment IDs

AssayMAP Protein Sample Prep Workbench User Guide

The software exports all the data for the selected experiment IDs to an *.expTags file, which is in XML format.

Importing experiment IDs

You can import the experiment IDs from a previously saved *.expTags file, for example as part of a data recovery process. However, you cannot import an experiment ID if it already exists in the Experiments database.

To import experiment IDs: 1 Do one of the following to open the Experiments Editor:

In an app or utility form, click Experiments Editor or Select Experiment. In the VWorks window, click Tools > Experiments Editor.

2 In the Experiments Editor window, click Import/Restore.

The Open dialog box appears.

2 Using Experiment IDs Exporting and importing experiment IDs

44 AssayMAP Protein Sample Prep Workbench User Guide

3 Select the file (*.expTags) that contains the experiment IDs to be imported, and then click Open. The Import/Restore Completed message appears and lists the import experiment IDs. The message also lists any experiment IDs that were not imported because they already exist in the Experiments database.

45

3 Affinity Purification v3.0 User Guide

This chapter contains the following topics: App description on page 46 Before you start on page 46 Preparing the solutions on page 51 Preparing the samples on page 55 Running the protocol on page 59 Assay development guidelines and protocol notes on page 68 Reference library on page 87 Note: This section presents instructions for using the Affinity Purification v3.0 application. If you are using the Aspiration Mode version, see Affinity Purification: Aspiration Mode v3.0 User Guide on page 89.

3 Affinity Purification v3.0 User Guide App description

46 AssayMAP Protein Sample Prep Workbench User Guide

App description

Affinity Purification v3.0. This application enables automated affinity purification of target molecules, such as antibodies and peptides, from 1 to 96 samples in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Affinity Purification protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

47

3 Affinity Purification v3.0 User Guide Before you start

AssayMAP Protein Sample Prep Workbench User Guide

Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. The following equipment is recommended when preparing the samples and reagents:

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

3 Affinity Purification v3.0 User Guide Before you start

48 AssayMAP Protein Sample Prep Workbench User Guide

Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Affinity Purification on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges:

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Protein A (PA-W) cartridge rack G5496-60000 G5496-60018

AssayMAP Protein G (PG-W) cartridge rack G5496-60008

AssayMAP Streptavidin (SA-W) cartridge rack G5496-60010 G5496-60021

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of Protein A, Protein G, or Streptavidin 5- or 25-L cartridges are required. For details, see Preparing the sample plates.

G5496-60009

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Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

PA-W, SA-W, and PG-W cartridges tolerate brief exposure to pH as low as 2.0. The stability of the PA-W, SA-W, and PG-W cartridges after capturing additional affinity ligands should be determined empirically.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware Manufacturer part number*

Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3, 5, 6, 7, 8

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Reagents

The volume, type, and concentration of reagents required for affinity purification vary depending on sample characteristics and the desired analytical result. Consult the resources that cover general principles of affinity purification such as those listed in the Reference library on page 87. For examples of reagents used with specific affinity interactions, consult the published scientific literature including publications that use the AssayMAP Bravo listed in the Agilent AssayMAP Bravo Citation Index. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure due to the buildup of salts within the syringe barrels. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide on page 567. All labware require volume overage for the protocol to execute properly. Use the Affinity Purification Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 51.

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 3, 5, 6, 7, 8

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 39

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7, 9**

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat Bottom, UV Star Greiner 675801 4, 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 3, 5, 6, 8

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner PCR plate is not compatible with the 25 L cartridges at deck locations 7 and 9.

Labware Manufacturer part number*

Deck location options

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3 Affinity Purification v3.0 User Guide Preparing the solutions

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Preparing the solutions

The following solutions are required for the Affinity Purification protocol: Priming & Equilibration Buffer Cartridge Wash Buffers Elution & Stringent Syringe Wash Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Affinity Purification

The Reagent Volume Calculator is a Microsoft Excel file that contains the following: Calculator worksheet. You enter the number of columns to process, whether to

perform the Collect Flow Through option, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

Reagent Recipe worksheet. You enter the concentrations of each component in your reagent, and the worksheet calculates the recipe volumes required.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

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5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figures show the worksheets of the Reagent Volume Calculator.

Figure Affinity Purification Calculator worksheet

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Figure Affinity Purification Reagent Recipe worksheet

Preparing the buffers

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the AssayMAP affinity purification chemistry.

Table Reagent preparation

Reagent (deck location) Composition and comments

Priming & Equilibration Buffer (deck location 3)

Typically a buffered aqueous solution with neutral pH and physiologic salt concentration, such as Phosphate-Buffered Saline (PBS), that is similar in composition to the buffer solution used to prepare the sample.

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Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and the cartridge positions in the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 55.

Cartridge Wash Buffer 1 (deck location 5)

High-stringency buffer (for example, a neutral buffer with high NaCl) or a low-stringency buffer (for example, PBS or a neutral mass-spec-friendly buffer) The Affinity Purification application is designed to allow the use of two wash buffers for high and low stringency washes or a single wash buffer. The buffer selection depends on a number of factors. First, consider whether Cartridge Wash Buffer 2 will or will not be used. This decision is often dependent on whether or not a stringent wash is desired in the protocol. If so, one would typically use the stringent buffer for Cartridge Wash Buffer 1 and then a less stringent wash buffer for Cartridge Wash Buffer 2, which would remove the stringent component of the buffer before elution. If a stringent buffer is not used, you would typically use Cartridge Wash Buffer 1 only, and it would be a low-stringency wash buffer, such as PBS or a neutral mass-spec-friendly buffer. Note: If you only do a single internal cartridge wash, it should be wash buffer 1, as skipping wash 1 and using wash 2 will result in skipping the sample chase which might decrease the amount of target recovered.

Cartridge Wash Buffer 2 (deck location 6)

Typically, a low-stringency buffer (for example PBS or a neutral mass- spec-friendly buffer) The composition of this buffer is often dictated by sensitivity of downstream steps to components of Wash Buffer 2 as the void volume in the cartridges (~2 L for 5 L cartridges or ~10 L for 25 L cartridges) will contain the last wash solution used before the Eluate step and end up in the eluate unless the Discard Eluate option is selected and this volume is discarded.

Elution & Stringent Syringe Wash Buffer (deck location 8)

12 mM HCl with 100 mM NaCl pH 2.0 or 5% acetic acid The elution solution is typically a low pH solution. A key consideration is if the sample will be neutralized following elution. If so, a 12 mM HCl solution with 100 mM NaCl is a good choice as it is very easy to neutralize. However, this solution requires approximately 46 column volumes to elute antibodies off the cartridge. If neutralization is not a key consideration or minimizing elution volume is a critical driver, a 5% acetic acid solution is a good choice because targets can be eluted in as little as 2 column volumes.

Reagent (deck location) Composition and comments

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To dispense the solutions into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Priming & Equilibration Buffer into the labware to be

placed at deck location 3. 3 Add the specified volume of Cartridge Wash Buffer 1 into the labware to be placed

at deck location 5. 4 If using two wash buffers, add the specified volume of Cartridge Wash Buffer 2 into

the labware to be placed at deck location 6. 5 Add the specified volume of Elution & Stringent Wash Buffer into the labware to be

placed at deck location 8. 6 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Affinity Purification protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to optimize the binding conditions (for example, pH). Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the sample composition

The optimal chemical environment for binding is generally similar for protein A, protein G, and streptavidin. Protein A and G resins bind selectively to antibodies.

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Examine the scientific literature for differences in their affinity for antibody subtypes from different species.

Streptavidin resin binds selectively to biotinylated molecules. Protein A, protein G, and streptavidin are relatively unaffected by most sample

components, including those present in complex protein mixtures.

What are optimal pH conditions? One of the most important considerations for optimizing binding conditions is the pH of the sample, which should be near neutral pH. Both low (2 to 3) and high (10 to 11) pH ranges can prevent binding to protein A or protein G resins. In general, the sample should be: Protein A. Greater than pH 6. Protein G. Greater than pH 4.

What sample components cause concerns? Protein A, protein G, and streptavidin generally tolerate moderate levels of salt, non- ionic detergents, and mild denaturing reagents, such as urea, quite well. You should examine scientific literature for the known effects and tolerances of protein A, protein G, and streptavidin, keeping in mind that these tolerances may differ depending on the antibody species and subtype.

Does the antibody species and isotype in the sample match the cartridge binding specificity? Protein A and protein G bind a wide variety of antibody subtypes and species (1). Carefully consider the species and subtype of antibody when choosing between using the AssayMAP Protein A or Protein G cartridge for purification. The antibodies that bind to protein G largely overlap the set that binds to protein A. While protein A is the industry standard for purification and titer determination of human therapeutic antibodies, protein G is the standard for purification of antibodies used as bioanalytical tools, primarily because many antibody subtypes are generated in species that bind poorly to protein A, for example, mouse IgG1 and rat IgG1 (2).

Determining the volume of sample to load

The AssayMAP Affinity Purification protocol permits loading up to 1000 L of sample onto AssayMAP cartridges. For sample volumes > 250 L, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 L) of the Bravo 96AM Head.

What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity: Quantitative binding capacity. The maximum mass of the target molecule that can

bind to the cartridge in a single pass, where less than 10% of the load appears in the flow-through. This value is dependent on the sample load flow rate.

Total binding capacity. The maximum mass of the target molecule that can bind to the cartridge. This can only be achieved by loading significantly more of the target molecule than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity.

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for Protein A, Protein G, and Streptavidin cartridges.

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What is the concentration of the target in the sample? If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

Does the experiment require quantitative binding of the target? For quantitative recovery of the target, the volume loaded must contain a mass of target protein that is equal to or less than the quantitative binding capacity. A bioprocess feed stream containing 10 mg/mL (10 g /L) antibody would require a maximum load volume of 10 L (100 g) to be within the quantitative range for 5 L PA-W cartridges. If the antibody concentration is unknown, you can do multiple runs at different volumes (10, 100, and 1000 L) to find a sample volume that is within the quantitative binding capacity, or a single run with the undiluted sample and multiple dilutions (no dilution, 1:10, and 1:100).

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows. The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use AssayMAP Resin-Free cartridges to fill the empty well positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash steps.

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Figure Examples of sample microplate and reservoir layout: A) Multiple of 8 samples, B) Not a multiple of 8

See Labware on page 49 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

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Running the protocol

The Affinity Purification protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the analyte from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the Cartridge & Tip Seating Station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 51. Prepare the samples. See Preparing the samples on page 55. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs. Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide on page 574. Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge

Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 48.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Affinity Purification application.

To set up the protocol: 1 Open the App Library. 2 Locate Affinity Purification, and then click App.

The Affinity Purification application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 65. To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

9 To save the method:

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a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 68.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

Equilibrate Aspirates Equilibration Buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates samples (deck location 4) into the syringes, and then dispenses them through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 100 5 3

25 L: 100 5 3

Range: 01000 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the sample flow-through at the wash station (deck location 1).

Cup Wash 1 Rinses the cartridge cups with Cartridge Wash Buffer 1 (deck location 5), and then discards the liquid into the wash station (deck location 1).

5 L: 25 3

25 L: 25 3

Range: 0100 010

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Internal Cartridge Wash 1

Aspirates Cartridge Wash Buffer 1 (deck location 5) into the syringes, and then dispenses it through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash 1 flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the Internal Cartridge Wash flow-through at the wash station (deck location 1).

Cup Wash 2 Rinses the cartridge cups with Cartridge Wash Buffer 2 (deck location 6) and discards the liquid into the wash station (deck location 1).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash 2

Aspirates Cartridge Wash Buffer 2 (deck location 6) into the syringes, and then dispenses it through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash 2 flow-through at the Flow Through Collection plate (deck location 7). If not selected, discards the Internal Cartridge Wash 2 flow-through at the wash station (deck location 1).

Stringent Syringe Wash

Aspirates Syringe Wash Buffer (deck location 8) into the syringes, and then discards the liquid into the wash station (deck location 1).

5 L: 50 2

25 L: 50 2

Range: 0250 010

Elute Aspirates Elution Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Eluate Collection (deck location 9).

5 L: 25 5 1

25 L: 125 5 1

Range: 0250 0.1500 010

Eluate Discard

If selected, a specified initial volume of Eluate will be dispensed through the cartridges, and collected in the Flow Through Collection plate (deck location 7), or discarded at the wash station (deck location 1).

5 L: 0

25 L: 0

Range: 0250

Add to Flow Through

If selected, collects the Eluate Discard in the Flow Through Collection plate (deck location 7). If not selected, discards the Eluate Discard at the wash station (deck location 1).

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner PCR plates are not compatible with the 25 L cartridges at deck locations 7 and 9.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

Existing Collection Volume

Specifies the volume of liquid present in the Eluate Collection plate (deck location 9) at the beginning of the run.

5 L: 0

25 L: 0

Range: 01000

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 68. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 82.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

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To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

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To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Affinity Purification protocol

to your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply

too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

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Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Prime This step removes entrained air from the packed resin bed and properly wets the surface of the resin. In preparation for priming, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP affinity purification cartridges (PA-W, SA-W, and PG-W) typically used with this application contain affinity ligands (proteins) covalently immobilized onto resin. These cartridge types should be primed with aqueous buffers containing no or low amounts of organic solvent or known protein denaturants. Because the Equilibration Buffer is drawn from the same reservoir as the Priming Buffer, buffers that favor analyte binding with minimal non-specific binding should be used for both priming and equilibration. This step is selected by default. Volume (L). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the resin bed. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes, and then performs an external syringe wash at the wash station to remove any sample remaining on the outside of the probes before mounting the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. The protocol accommodates sample volumes up to 1000 L to be dispensed through the AssayMAP affinity purification cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L. Each syringe has a maximum capacity of 250 L. When sample volumes are greater than 250 L, the protocol will iteratively load samples onto cartridges. To determine the number and volume of iterative load steps, the protocol uses the following formulas: # of times to load = total sample volume/250,

where # times to load is rounded up to nearest integer volume of each load = sample volume/# of times to load For example, if the total sample volume is 900 L, then: # times to load = 900/250 = 3.6, which is rounded up to 4 volume of each load = 900/4 = 225 If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

IMPORTANT Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For more information, see Preparing the sample plates on page 57.

To determine the volume of sample to load, see Determining the volume of sample to load on page 56. This step is selected by default.

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Volume (L). The volume of sample to load should be balanced with the sample concentration and the mass capacity of the cartridge. Default: 100 Practical: 101000 Range: 01000 Note: The lower the sample volume, the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed into the wash station. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.

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Cup Wash 1 This step removes the residual sample solution that may remain above the resin bed after the Load Samples step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 1

This step uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the resin bed. In preparation for Internal Cartridge Wash 1, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Cartridge Wash Buffer 1 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 1 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. If the Load Samples step is selected, the first 5 L (5 L cartridges) or 25 L (25 L cartridges) of Cartridge Wash Buffer 1 is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash 1 volume minus the chase volume is dispensed at the Internal Cartridge Wash 1 flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the flow-through from Internal Cartridge Wash 1 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed into the wash station. This step is not selected by default.

Cup Wash 2 This step removes the residual buffer that may remain above the resin bed after the Internal Cartridge Wash 1 step. This step aspirates Cartridge Wash Buffer 2 and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the previous cartridge wash is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are parked at the seating station, and then the liquid in the syringes is dispensed into the wash station. The syringes are washed at the wash station. This step is selected by default. Volume (L). A volume less than the default may be insufficient for cup washing, while a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 2

This step uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules and Cartridge Wash Buffer 1 from the resin bed. In preparation for Internal Cartridge Wash 2, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid that is in the cups is removed by a 60 L aspiration, the aspirated solution is discarded at the wash station, 10 L of Cartridge Wash Buffer 2 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 2 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer from the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but will also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to 0 skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the liquid eluted during Internal Cartridge Wash 2 is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through is dispensed into the wash station. Select this step if you want to collect the flow-through generated during Internal Cartridge Wash 2. This step is not selected by default.

Stringent Syringe Wash

This step cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the wash station. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

Elute This step uses Elution Buffer to elute bound analytes from the cartridges. In preparation for elution, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the eluate is mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 L, the samples will not be mixed. You can also select the Eluate Discard and Add to Flow Through substeps, which are described in the following rows of this table.

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Elute (continued) This step is selected by default. Volume (L). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges, or 5075 L for 25 L cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Note: The Eluate Collection plate must be able to accommodate the total volume, which is determined by summing the net elution volume (Elute volume - Eluate Discard volume) and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Volume for 5 L cartridges:

Default: 25 Practical: 1030 Range: 0250

Volume for 25 L cartridges: Default: 125 Practical: 50150 Range: 0250

Note: Setting the volume to zero skips all Elute tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/ min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500 Wash cycle. The number of syringe washes to perform at the wash station after an Elute step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Eluate Discard This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. If the Eluate Discard step is selected, the specified volume is dispensed into the wash station or Flow Through Collection plate (if the Add to Flow Through step is selected). The remaining Elution Buffer is dispensed through cartridges into the Eluate Collection plate.

Example: If the Elute, Eluate Discard, and Add to Flow Through steps are all selected with the following settings: Elute volume = 15 L (5 L cartridges) or 40 L (25 L cartridges) Eluate Discard volume = 2 L (5 L cartridges) or 10 L (25 L cartridges) the first 2 L (5 L cartridges) or 10 L (25 L cartridges) eluate from the cartridges will be discarded into the Flow Through Collection plate, and the remaining 13 L (5 L cartridges) or 30 L (25 L cartridges) eluate will be collected in the Eluate Collection plate.

Select the Eluate Discard step in situations where minimizing the volume of eluate is important. For AssayMAP cartridges, the initial elution volume (~2 L for 5 L cartridges and ~10 L for the 25 L cartridges) contains small or no measurable amounts of analyte. This option is not selected by default. Volume (L). The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. Default: 0 Practical:

5 L cartridges: 02 25 L cartridges: 010

Range: 0250

Add to Flow Through

If selected, this step dispenses the Eluate Discard volume into the Flow Through Collection plate. If the Add to Flow Through step is not selected, the Eluate Discard is dispensed into the wash station. This step is not selected by default. Note: The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected.

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Existing Collection Volume

This step enables you to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. The Existing Collection Volume and the net volume from the Elute step (Elute volume - Eluate Discard volume) feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head into and out of the wells during elution and eluate mixing in a volume- dependent manner. For the maximum practical working volumes of labware for eluate collection, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, for adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (L): Default: 0 Practical: 0250 Range: 01000 Note: Total elution collection well volumes above 500 L may require additional off- deck mixing to reach homogeneity.

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 L of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Affinity Purification protocol using the default method settings. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Start protocol 2 Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes.

Prime 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then does an external probe wash.

3 Aspirates 10 L of Priming Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

3 Aspirates the Priming Buffer.

2 Mounts the cartridges on the head.

1 Dispenses the Priming Buffer through the cartridges into the wash station between the chimneys, and then does an external cartridge wash.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

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Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then does an external probe wash.

3 Aspirates 10 L of Equilibration Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Equilibration Buffer into the cartridge cups and exercises the cartridges off task.

3 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

1 Dispenses the Equilibration Buffer through the cartridges to equilibrate, and then does an external cartridge wash.

2 Parks the cartridges in the seating station.

1 Washes the syringes at the wash station.

Load Samples 4 Aspirates samples into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

7 Dispenses the samples through the cartridges and into the Flow Through Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 1 5 Aspirates Cartridge Wash Buffer 1 into the syringes.

2 Washes the cartridge cups and exercises the cartridges off task.

1 Dispenses the buffer into the wash station between the chimneys.

1 Washes the syringes.

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Action

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Internal Cartridge Wash 1

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then does and external probe wash.

5 Aspirates 10 L of Cartridge Wash Buffer 1 for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

5 Aspirates Cartridge Wash Buffer 1 into the syringes for the sample chase and Internal Cartridge Wash 1 steps.

2 Mounts the cartridges on the head.

1 Dispenses 5 L (5 L cartridges) or 25 L (25 L cartridges) Cartridge Wash Buffer 1 through the cartridges at the Load Samples flow rate for the sample chase step.

1 Dispenses the remaining Cartridge Wash Buffer 1 through the cartridges at the Internal Cartridge Wash 1 flow rate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 2 6 Aspirates Cartridge Wash Buffer 2 into the syringes.

2 Washes the cartridge cups and exercises the cartridges off task.

1 Dispenses buffer into the wash station between the chimneys.

1 Washes the syringes.

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Internal Cartridge Wash 2

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then does an external probe wash.

6 Aspirates 10 L of Cartridge Wash Buffer 2 for the cartridge air-gap prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates Cartridge Wash Buffer 2 into the syringes.

2 Mounts the cartridges on the head.

1 Dispenses Cartridge Wash Buffer 2 through the cartridges.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Stringent Syringe Wash

8 Aspirates the Stringent Syringe Wash Buffer (Elution Buffer).

1 Dispenses the buffer at the wash station.

1 Washes the syringes.

Elute 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then does an external probe wash.

8 Aspirates 10 L of Elution Buffer for the cartridge air-gap-prevention step.

2 Dispenses the 10 L of Elution Buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Elution Buffer.

2 Mounts the cartridges on the head.

9 Elutes the samples into the Eluate Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges at the seating station.

9 Mixes eluates.

1 Washes the syringes.

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Final Syringe Wash 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys.

1 Washes the syringes.

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Action

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Reference library

1 Terry, M.P. in Handbook of Affinity Chromatography, Second Edition. (ed. D.S. Hage), 367-397 (CRC Press, 2005).

2 PROTEUS Protein G Antibody Purification Handbook, Mini & Midi spin columns, Bio- Rad Laboratories, Inc 2013.

See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Bravo Platform.

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This chapter contains the following topics: App description on page 90 Before you start on page 90 Preparing the solutions on page 95 Preparing the samples on page 98 Running the protocol on page 102 Assay development guidelines and protocol notes on page 110 Reference library on page 124 Note: This section presents instructions for using the Affinity Purification: Aspiration Mode v3.0 application. If you are using the Dispense Mode version, see Affinity Purification v3.0 User Guide on page 45.

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App description

Affinity Purification: Aspiration Mode v3.0. This application enables automated affinity purification of target molecules, such as antibodies and peptides, from 1 to 96 samples in a single run. This application aspirates the sample and wash solutions up through the cartridge resin bed rather than dispensing them through the resin bed, which is how the standard Affinity Purification application functions. For most customers, Agilent recommends using the standard Affinity Purification application instead of the Affinity Purification: Aspiration Mode application. Although the two applications yield similar results, the standard Affinity Purification application is less sensitive to clogging and, therefore, more robust. However, some customers find the Affinity Purification: Aspiration Mode application provides slightly better purification.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Affinity Purification: Aspiration Mode protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Note: The 25-L cartridges have not yet been optimized on the Affinity Purification: Aspiration Mode application. If you are interested in trying them on this application, contact Agilent Customer Service for advice.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Optional equipment. The following equipment is recommended when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Affinity Purification on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges. Note: This application has not yet been optimized for the 25 L cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

AssayMAP Protein A (PA-W) cartridge rack G5496-60000

AssayMAP Protein G (PG-W) cartridge rack G5496-60008

AssayMAP Streptavidin (SA-W) cartridge rack G5496-60010

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of Protein A, Protein G, or Streptavidin cartridges are required. For details, see Preparing the sample plates.

G5496-60009

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Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges: Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

PA-W, SA-W, and PG-W cartridges tolerate brief exposure to pH as low as 2.0. The stability of the PA-W, SA-W, and PG-W cartridges after capturing additional affinity ligands should be determined empirically.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

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Reagents

The volume, type, and concentration of reagents required for affinity purification vary depending on sample characteristics and the desired analytical result. Consult the resources that cover general principles of affinity purification such as those listed in the Reference library on page 124. For examples of reagents used with specific affinity interactions, consult the published scientific literature including publications that use the AssayMAP Bravo which can be found in the Agilent AssayMAP Bravo Citation Index. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure due to the buildup of salts within the syringe barrels. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide on page 567. All labware require volume overage for the protocol to execute properly. Use the Affinity Purification Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 95.

Labware Manufacturer part number*

Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 39

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 39

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 39

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 39

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 675801, Half Area, Flat Bottom, UV Star Greiner 675801 39

96 V11 Manual Fill Reservoir Agilent G5498B#049 39

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Preparing the solutions

The following solutions are required for the Affinity Purification: Aspiration Mode protocol: Priming & Equilibration Buffer Cartridge Wash Buffers Elution & Stringent Syringe Wash Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Affinity Purification: Aspiration Mode

The Reagent Volume Calculator is a Microsoft Excel file that contains the following: Calculator worksheet. You enter the number of columns to process, whether to

perform the Collect Flow Through option, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

Reagent Recipe worksheet. You enter the concentrations of each component in your reagent, and the worksheet calculates the recipe volumes required.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click Restore Defaults. 5 Modify the values in the green boxes as required to match your specific method. As

you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red.

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To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figures show the worksheets of the Reagent Volume Calculator.

Figure Affinity Purification: Aspiration Mode Calculator worksheet

Figure Reagent Recipe worksheet

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Preparing the buffers

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the AssayMAP affinity purification chemistry.

Table Reagent preparation

Reagent and deck location Composition and comments

Priming & Equilibration Buffer (deck location 3)

Typically a buffered aqueous solution with neutral pH and physiologic salt concentration, such as Phosphate-Buffered Saline (PBS), that is similar in composition to the buffer solution used to prepare the sample.

Cartridge Wash Buffer 1 (deck location 5)

High-stringency buffer (for example, a neutral phosphate buffer with high NaCl) or a low-stringency buffer (for example, PBS or a neutral mass-spec- friendly buffer) The Affinity Purification: Aspiration Mode application is designed to allow the use of two wash buffers for high- and low-stringency washes or a single wash buffer. The buffer selection depends on a number of factors. First, consider whether Cartridge Wash Buffer 2 will or will not be used. This decision is often dependent on whether or not a stringent wash is desired in the protocol. If so, one would typically use the stringent buffer for Cartridge Wash Buffer 1 and then a less stringent wash buffer for Cartridge Wash Buffer 2, which would remove the stringent component of the buffer before elution. If a stringent buffer is not used, you would typically use Cartridge Wash Buffer 1 only, and it would be a low-stringency wash buffer, such as PBS or a neutral mass-spec-friendly buffer. Note: If you only do a single internal cartridge wash, it should be wash buffer 1, as skipping wash 1 and using wash 2 will result in skipping the sample chase which might decrease the amount of target recovered.

Cartridge Wash Buffer 2 (deck location 6)

Typically, a low-stringency buffer (for example PBS or a neutral mass- spec-friendly buffer) The composition of this buffer is often dictated by sensitivity of downstream steps to components of Cartridge Wash Buffer 2 as the void volume in the cartridges (~2 L for 5 L cartridges or 10 L for 25 L cartridges) will contain the last wash solution used before the Eluate step and end up in the eluate unless the Discard Eluate option is selected and this volume is discarded.

Elution & Syringe Wash Buffer (deck location 8)

12 mM HCl with 100 mM NaCl pH 2.0 or 5% acetic acid The elution solution is typically a low pH solution. A key consideration is if the sample will be neutralized following elution. If so, a 12 mM HCl solution with 100 mM NaCl is a good choice as it is very easy to neutralize. However, this solution approximately 46 column volumes to elute antibodies off the cartridge. If neutralization is not a key consideration or minimizing elution volume is a critical driver, a 5% acetic acid solution is a good choice because targets can be eluted in as little as 2 column volumes.

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Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and cartridge positions on the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 98.

To dispense the reagents into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Priming & Equilibration Buffer into the labware to be

placed at deck location 3. 3 Add the specified volume of Cartridge Wash Buffer 1 into the labware to be placed

at deck location 5. 4 If using two wash buffers, add the specified volume of Cartridge Wash Buffer 2 into

the labware to be placed at deck location 6. 5 Add the specified volume of Elution & Syringe Wash Buffer into the labware to be

placed at deck location 8. 6 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Affinity Purification: Aspiration Mode protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to optimize the binding conditions (for example, pH). Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

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Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the sample composition

The optimal chemical environment for binding is generally similar for protein A, protein G, and streptavidin. Protein A and G resins bind selectively to antibodies.

Examine the scientific literature for differences in their affinity for certain antibody subtypes from different species.

Streptavidin resin binds selectively to biotinylated molecules. Protein A, protein G, and streptavidin are relatively unaffected by most sample

components, including those present in complex protein mixtures.

What are optimal pH conditions? One of the most important considerations for optimizing binding conditions is the pH of the sample, which should be near neutral pH. Both low (2 to 3) and high (10 to 11) pH ranges can prevent binding to protein A or protein G resins. In general, the sample should be: Protein A. Greater than pH 6. Protein G. Greater than pH 4.

What sample components cause concerns? Protein A, protein G, and streptavidin generally tolerate moderate levels of salt, non-ionic detergents, and mild denaturing reagents, such as urea, quite well. You should examine scientific literature for the known effects and tolerances of protein A, protein G, and streptavidin, keeping in mind that these tolerances may differ depending on the antibody species and subtype.

Does the antibody species and isotype in the sample match the cartridge binding specificity? Protein A and protein G bind a wide variety of antibody subtypes and species (1). Carefully consider the species and subtype of antibody when choosing between using the AssayMAP Protein A or Protein G cartridge for purification. The antibodies that bind to protein G largely overlap the set that binds to protein A. While protein A is the industry standard for purification and titer determination of human therapeutic antibodies, protein G is the standard for purification of antibodies used as bioanalytical tools, primarily because many of these antibody subtypes are generated in species that bind poorly to protein A, for example, mouse IgG1 and rat IgG1 (2).

Determining the volume of sample to load

The AssayMAP Affinity Purification: Aspiration Mode protocol permits loading up to 1000 L of sample onto AssayMAP cartridges. For sample volumes > 250 L, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 L) of the Bravo 96AM Head.

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What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity: Quantitative binding capacity. The maximum mass of the target molecule that can

bind to the cartridge in a single pass, where less than 10% of the load appears in the flow-through. This value is dependent on the sample load flow rate.

Total binding capacity. The maximum mass of the target molecule that can bind to the cartridge. This can only be achieved by loading significantly more of the target molecule than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity.

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for Protein A, Protein G, and Streptavidin cartridges.

What is the concentration of the target in the sample? If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

Does the experiment require quantitative binding of the target? For quantitative recovery of the target, the volume loaded must contain a mass of target protein that is equal to or less than the quantitative binding capacity. A bioprocess feed stream containing 10 mg/mL (10 g /L) antibody would require a maximum load volume of 10 L (100 g) to be within the quantitative range for 5 L PA-W cartridges. If the antibody concentration is unknown, you can do multiple runs at different volumes (10, 100, and 1000 L) to find a sample volume that is within the quantitative binding capacity, or a single run with the undiluted sample and multiple dilutions (no dilution, 1:10, and 1:100).

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows. The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use AssayMAP Resin-Free cartridges to fill the well positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash steps.

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Figure Example of sample microplate and reservoir layout: A) Multiple of 8 samples, and B) Not a multiple of 8

See Labware on page 93 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

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Running the protocol

The Affinity Purification: Aspiration Mode protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the analyte from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the Cartridge & Tip Seating Station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 95. Prepare the samples. See Preparing the samples on page 98.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v3.0 User Guide.

Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 93.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Affinity Purification: Aspiration Mode application.

To set up the protocol: 1 Open the App Library. 2 Locate Affinity Purification: Aspiration Mode, and then click App.

The Affinity Purification: Aspiration Mode application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 108. To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Verify that the Number of box selection is 5L Cartridges. b In the box, type the number of cartridges present in the cartridge holder at deck

location 2. The position of the cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 896 Default: 8

IMPORTANT Ensure that you specify the number of cartridges for the Affinity Purification: Aspiration Mode app instead of the number of columns, which is the setting used in the other AssayMAP apps. Also, make sure to use a column format for the microplate setup to avoid droplets falling on the deck as described in Planning the microplate setup on page 100.

CAUTION If the cartridge selection is greater than the actual number of cartridges used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the form specifies 96 cartridges, but only 8 cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the cartridge selection is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the form specifies 8 cartridges, but 96 cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. To prevent potential equipment damage, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings area, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

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9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 110.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

Range: 010

Prime Aspirates the Priming & Equilibration buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 100 300

Range: 100 300

Equilibrate Dispenses the Priming & Equilibration buffer through the cartridges into the wash station (deck location 1).

5 L: 50 10

Range: 0140 0.5500

Load Samples

Aspirates up to 245 L of samples (deck location 4) through the mounted cartridges into the syringes, performs an external cartridge tip wash at the wash station (deck location 1), and then aspirates a 5- L chase of Equilibration Buffer (deck location 3). The cartridges are removed (deck location 2) and then the flow-through is dispensed into either Flow Through Collection (deck location 7) or the wash station (deck location 1). Samples >245 L are loaded in multiple steps.

5 L: 100 5 3

Range: 01000 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through at the Flow Through Collection (deck location 7). If not selected, discards the sample flow-through at the wash station (deck location 1).

Cup Wash 1 Rinses the cartridge cups with Cartridge Wash Buffer 1 (deck location 5), and then discards the liquid into the wash station (deck location 1).

5 L: 25 3

Range: 0100 010

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

Internal Cartridge Wash 1

Aspirates Cartridge Wash Buffer 1 (deck location 5) through the mounted cartridges, removes the cartridges from the probes, and dispenses the contents of the syringes into the wash station (deck location 1).

5 L: 50 10 3

Range: 0250 0.5500 010

Cup Wash 2 Rinses the cartridge cups with Cartridge Wash Buffer 2 (deck location 6) and discards the liquid into the wash station (deck location 1).

5 L: 25 3

Range: 0100 010

Internal Cartridge Wash 2

Aspirates Cartridge Wash Buffer 2 (deck location 6) through the mounted cartridges, removes the cartridges from the probes, and dispenses the contents of the syringes into the wash station (deck location 1).

5 L: 50 10 3

Range: 0250 0.5500 010

Stringent Syringe Wash

Aspirates Syringe Wash Buffer (deck location 8) into the syringes, and then discards the liquid into the wash station (deck location 1).

5 L: 50 2

Range: 0250 010

Elute Aspirates Elution Buffer (deck location 8) into the syringes, and then dispenses the buffer through the cartridges into the Eluate Collection plate (deck location 9).

5 L: 25 5

Range: 0250 0.1500

Re- Equilibrate

Aspirates Equilibration Buffer (deck location 3) through the mounted cartridges into the syringes (aspirate mode), removes the cartridges, and then dispenses liquid into the wash station (deck location 1).

5 L: 50 10

Range: 0250 0.5500

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 111. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 121.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

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To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

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c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Affinity Purification:

Aspiration Mode protocol to your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

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Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that this selection is correct. If the cartridge selection is: Greater than the actual number used, the Bravo Platform will apply too much force

when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the form specifies 96 cartridges, but only 8 cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 8 cartridges, but 96 cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

IMPORTANT Although you specify the number of cartridges for this app, you specify the number of columns of cartridges for other apps.

Default: 8 Range: 1-12

Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

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Prime This step removes entrained air from the packed resin bed and properly wets the surface of the resin. The Prime step aspirates enough Priming Buffer into the syringes to wet the inlet seal (10 L), and perform the Prime (100 L) and Equilibration (50 L default) steps. In preparation for priming, 10 L of Priming Buffer is dispensed into each cartridge cup to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. After mounting the cartridges on the probes, the Priming Buffer is dispensed (100 L at 300 L/min) through the cartridges into the wash station. The AssayMAP affinity purification cartridges (PA-W, SA-W, and PG-W) typically used with this application contain proteins covalently immobilized onto resin. These cartridge types should be primed with aqueous buffers containing no or low amounts of organic solvent or known protein denaturants. Because the Equilibration Buffer is drawn from the same reservoir as the Priming Buffer, buffers that favor analyte binding should be used for both priming and equilibration. This step is selected by default. Volume (L). The volume is sufficient to wet and remove entrained air from the resin bed. Default: 100 Range: 100 Flow rate (L/min). Default: 300 Range: 300

Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. This step dispenses the Equilibration Buffer remaining in the syringes through the cartridges. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Default: 50 Practical: 50100 Range: 0140 If you select Prime and Equilibrate, the system calculates the volume required to prewet the cartridges and perform both steps and then aspirates that volume during the Prime step. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500

Protocol step Guidelines and notes

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. This step mounts the cartridges on the syringe probes, aspirates the sample through the resin bed, and washes the exterior of the cartridge tips at the wash station to remove any sample on the outside of the cartridge tips. A 5 L sample chase of Priming & Equilibration Buffer is aspirated through the resin bed at the same flow rate as the sample load to ensure that the sample in the resin bed at the end of the sample load has the same residence time for binding as the rest of the sample. An exterior cartridge wash is performed, cartridges are ejected, and then the contents in the syringe (flow- through) are deposited into the Flow Through Collection plate or the wash station. The protocol accommodates sample volumes up to 1000 L to be aspirated through the AssayMAP affinity purification cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L. Each syringe has a maximum capacity of 250 L. When sample volumes are greater than 250 L, the protocol will iteratively load samples onto cartridges in equal volume steps. To determine the number of times to load and the volume of each iterative load, the protocol uses the following formulas:

# of times to load = total sample volume/250, where # times to load is rounded up to nearest integer volume of each load = sample volume/# of times to load

For example, if the total sample volume is 900 L, then: # times to load = 900/250 = 3.6, which is rounded up to 4 volume of each load = 900/4 = 225

If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

IMPORTANT Be sure to include the recommended labware-dependent volume overage to prevent air from entering the cartridge. For more information, see Preparing the sample plates on page 100.

This step is selected by default. Volume (L). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. To determine the volume of sample to load, see Determining the volume of sample to load on page 99. Default: 100 Practical: 101000 Range: 01000 Note: The lower the sample volume the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing.

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Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. Default: 5 Practical: 215 Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed into the wash station. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is not selected by default. Note: A constant 5-L volume is used as a sample chase during the Load Samples step, which results in some dilution of sample flow-through. For example, if you load a 50-L sample, the flow-through will contain the volume of the sample plus 5 L of sample chase. Make sure you consider this volume when calculating the flow-through concentration.

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Cup Wash 1 This step removes the residual sample solution that may remain above the resin bed after the Load Samples step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 1

This step uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the resin bed. For the wash operation, this step aspirates Cartridge Wash Buffer 1 through the mounted cartridges into the syringes, parks the cartridges at the seating station, and then dispenses the syringe contents into the wash station at an offset from the chimneys. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Cup Wash 2 This step removes the residual buffer that may remain above the resin bed after the Internal Cartridge Wash 1 step. This step aspirates Cartridge Wash Buffer 2 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the previous cartridge wash is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are parked at the seating station, and then the liquid in the syringes is dispensed into the wash station. The syringes are washed at the wash station. This step is selected by default. Volume (L). A volume less than the default may be insufficient for cup washing, while a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 2

This step uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules and Cartridge Wash Buffer 1 from the resin bed. For the wash operation, this step aspirates Cartridge Wash Buffer 2 through the mounted cartridges into the syringes, parks the cartridges at the seating station, and then dispenses the buffer into the wash station at an offset from the chimneys. The syringes are washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but will also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to 0 skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Stringent Syringe Wash

This step cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the wash station. The syringes are then washed at the wash station. This step is selected by default. Volume (L/min). Volumes higher than the default volume may improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

Elute This step uses Elution Buffer to elute bound analytes from the cartridges. This step aspirates the buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges. This step is selected by default. Volume (L). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges). The default volume is conservative and significantly higher than the minimum expected with a strong Elution Buffer. Default: 25 Practical: 1030 Range: 0250 Flow rate (L/min). A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500

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Re-Equilibrate Uses the Equilibration Buffer to return the cartridge to a neutral pH solution. During the Re-Equilibrate step, the Equilibration Buffer is aspirated through the mounted cartridges into the syringes. The cartridges are parked at the seating station and then the liquid is dispensed into the wash station between the chimneys. The syringes are washed, and then the syringes are used to mix the samples in the Eluate Collection plate. Note: If the total volume in the Eluate Collection plate is <15 L, the samples are not mixed. This step is selected by default. Note: If the Re-Equilibrate step is skipped, the eluate will not be mixed. Volume (L). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Equilibrate tasks. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. During each Final Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

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Minimizing the duration of your assay

To minimize the duration of your assay: If the sum of the sample load, chase, and both internal cartridge washes is less than 250 L, modify the following protocol settings to minimize the Bravo Platform actions: Load Samples: Set the number of wash cycles to 0. Collect Flow Through: Not selected. Cup Wash 1: Not selected. Internal Cartridge Wash 1: Set the number of wash cycles to 0. (If Internal Cartridge

Wash 2 is not selected, set the number of wash cycles to any desired value.) Cup Wash 2: Not selected. These settings result in accumulation of the flow-through, chase, and any internal cartridge wash liquids in the syringe without requiring removal of the cartridges from the probes. This total volume must be less than the 250 L syringe capacity, as the cartridges will dismount for larger accumulated volumes and introduce the potential for contamination of the cup, which would make skipping the cup washes a poor choice. The software keeps track of cumulative volume of the sample (which equals the flow- through), 5-L chase, and Internal Cartridge Wash 1 and 2 drawn into the syringe, and performs the steps required to empty the syringe when it reaches the 250-L capacity. As long as the accumulated volume is less than 250 L, this volume can be held in the syringes until the Stringent Syringe Wash initiates.

Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Affinity Purification: Aspiration Mode protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Start protocol 2 Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes the specified number of times.

Prime 3 Aspirates the Priming & Equilibration Buffer for the air-gap prevention and the Prime and Equilibrate steps.

2 Dispenses 10 L of buffer into each cartridge cup for air-gap prevention.

2 Mounts the cartridges on the syringe probes.

1 Dispenses the buffer for the Prime step through the cartridges into the wash station at the priming flow rate.

Equilibrate 1 Dispenses the remaining buffer through the cartridges into the wash station at the Equilibration flow rate, and then washes the exterior of the cartridges.

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Load Samples 4 Aspirates samples through the mounted cartridges into the syringes.

1 Washes the exterior of the cartridges.

3 Aspirates a 5 L of Priming & Equilibration Buffer for the sample chase.

1 Washes the exterior of the cartridges.

2 Parks the cartridges at the seating station.

1 Dispenses the sample flow-through into the wash station.

1 Washes the syringes.

Cup Wash 1 5 Aspirates Cartridge Wash Buffer 1 into the syringes.

2 Performs the cup wash and exercises the cartridges off task.

1 Dispenses the buffer at the wash station.

1 Washes the syringes at the wash station.

Internal Cartridge Wash 1

2 Mounts the cartridges on the syringe probes.

5 Aspirates Cartridge Wash Buffer 1 through the mounted cartridges.

1 Washes the exterior of the cartridges.

2 Parks the cartridges at the seating station.

1 Dispenses the buffer into the wash station.

1 Washes the syringes.

Cup Wash 2 6 Aspirates Cartridge Wash Buffer 2 into the syringes.

2 Performs the cup wash and exercises the cartridges off task.

1 Dispenses the buffer at the wash station.

1 Washes the syringes at the wash station.

Internal Cartridge Wash 2

2 Mounts the cartridges on the syringe probes.

6 Aspirates Cartridge Wash Buffer 2 through the mounted cartridges.

1 Washes the exterior of the cartridges.

2 Parks the cartridges at the seating station.

1 Dispenses the buffer into the wash station.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Stringent Syringe Wash

8 Aspirates Syringe Wash Buffer.

1 Dispenses the buffer into the wash station.

1 Washes the exterior of the syringes.

Elute 8 Aspirates the Elution Buffer.

2 Mounts the cartridges on the syringe probes.

9 Elutes the samples into the Eluate Collection plate.

1 Washes the exterior of the cartridge tips.

Re-Equilibrate and Eluate Mix

3 Aspirates Equilibration Buffer through the cartridges into the syringes.

2 Parks the cartridges at the seating station.

1 Dispenses the buffer, and then washes the syringes.

9 Mixes the samples in the Eluate Collection plate using the syringes.

Final Syringe Wash 1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Reference library

1 Terry, M.P. in Handbook of Affinity Chromatography, Second Edition. (ed. D.S. Hage), 367-397 (CRC Press, 2005).

2 PROTEUS Protein G Antibody Purification Handbook, Mini & Midi spin columns, Bio- Rad Laboratories, Inc 2013.

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This chapter contains the following topics: App description on page 126 Before you start on page 126 Preparing the solutions on page 131 Preparing the samples on page 133 Running the protocol on page 136 Assay development guidelines and protocol notes on page 145 Reference library on page 158

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App description

Fractionation v2.0. This application enables automated stepwise elution of samples in up to six fractions using different stringency solutions, such as buffers with increasing ionic strength, increasing pH, or increasing organic concentration.

Before you start

This topic lists the required hardware, software, AssayMAP kits, cartridges, labware, and reagents for running the Fractionation protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. The following equipment is recommended when preparing the samples and reagents:

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Microplate centrifuge, such as the Agilent Microplate Centrifuge Microplate vacuum concentrator for concentration and drying of samples Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

Starter kit, cartridges, and labware

The starter kit for the Fractionation application contains both strong cation exchange (SCX) cartridges and labware.

AssayMAP cartridges The following table lists the available AssayMAP cartridges that are commonly used to perform fractionation on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Starter kit Agilent Part number

AssayMAP SCX Fractionation Starter Kit Contains 96 strong cation exchange (SCX) cartridges and Labware for the Fractionation protocol.*

G5496-60014

* The SCX Fractionation kit includes16x Greiner 650201_U-Bottom, Clear PolyPro and 1x 96 ABgene 1127, 1mL Deep Well, Square Well, Round bottom plate.

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP SCX cartridge rack (Qty 96) 5190-6536

AssayMAP C18 cartridge rack (Qty 96) 5190-6532 G5496-60017

AssayMAP RP-S cartridge rack (Qty 96) G5496-60033 G5496-60023

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Note: The C18 and RP-S cartridges contain reversed-phase resin. Agilent suggests using RP-S for high pH reversed-phase fractionation because it is more stable in high pH solutions. For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges: Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

Labware Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of SCX, C18, or RP-S 5- or 25-L cartridges are required. For details, see Preparing the sample plates on page 134.

G5496-60009

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

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Figure Labware locations on the Bravo deck (top view)

Reagents

The AssayMAP Fractionation protocol is designed to permit multiple types of fractionation to be performed. Therefore, the reagent requirements are dependent on the fractionation strategy and the chemistry of the AssayMAP cartridges. For examples, consult the published scientific literature, including publications that use the AssayMAP Bravo which can be found in the Agilent AssayMAP Bravo Citation Index. In general, buffer systems used for liquid chromatography separations in a column format can be adapted for use in the AssayMAP cartridge format. Fractionation using AssayMAP SCX cartridges can be accomplished using elution buffers with increasing ionic strength or increasing pH. Similarly, both C18 and RP-S cartridges can be used for high-pH, reversed-phase fractionation using increasing concentrations of organic solvent in the elution buffers. Because of the high-pH stability of RP-S cartridges, RP-S cartridges are recommended for high-pH, reversed-phase fractionation with increasing concentrations of organic solvent. All labware require volume overage for the protocol to execute properly. Use the Fractionation Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 131.

Labware Manufacturer part number*

Deck location options

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 3

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 49

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 49

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Preparing the solutions

The following solutions are required for the Fractionation protocol: Priming Buffer Equilibration and Wash Buffer Elution Buffers You can use the supplied Reagent Volume Calculator for the Fractionation application to facilitate recipe volume calculations.

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Fractionation

The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, whether to perform the Collect Flow Through options, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click Restore Defaults to set the values in the calculator using the values from the

default method for the 5 L cartridges. 5 Modify the values in the green boxes as required to match your specific method. As

you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red.

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To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figure shows the worksheet of the Reagent Volume Calculator.

Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and the cartridge positions in the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 133.

To dispense the reagents into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Priming Buffer into the labware to be placed at deck

location 6. 3 Add the specified volume of Equilibration & Wash Buffer into the labware to be

placed at deck location 9.

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4 Add the specified volume of Elution Buffer into the labware (16) to be stacked at deck location 4.

5 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Fractionation protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to optimize the binding conditions (for example, pH). Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the buffer composition

Depending on the type of fractionation you are performing the sample should be in a buffer compatible with the stationary phase. For peptide fractionation using SCX cartridges, optimum performance is typically achieved under low salt and low pH conditions. For high- or low-pH peptide fractionation using C18 and RP-S cartridges, samples typically should have a pH of < 3.

Determining the volume of sample to load

The AssayMAP Fractionation protocol permits loading up to 250 L of sample onto AssayMAP cartridges due to labware capacity limits.

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What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity: Quantitative binding capacity. The maximum mass of peptide that can bind to the

cartridge in a single pass, where less than 10% of the load appears in the flow- through. For reversed-phase cartridges, the quantitative binding capacity is relatively straightforward for a single species of peptide. The quantitative binding capacity for a mixture of peptides is more complex due to the differences in relative hydrophobicity of the peptides, which results in competitive binding in situations where the ratio of binding sites to mass loaded is low. For examples of how sample mass loaded and the relative hydrophobicity of peptides can affect recovery in a complex peptide mixture, see Agilent Application Note 5991-2957EN in the Reference library on page 158. To avoid or minimize sample bias during peptide cleanup, it is critical to load sample masses that are less than the mass at which hydrophillic peptides are lost. See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed quantitative cartridge binding capacity information for the 5 and 25 L RP-S and C18 cartridges.

Total binding capacity. The maximum mass of peptide that can bind to the cartridge. This can only be achieved by loading significantly more peptide than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity and will result in the loss of hydrophillic peptides. See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed total binding capacity.

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows. The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use AssayMAP Resin-Free cartridges to fill the empty well positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash steps.

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Figure Example of sample microplate and reservoir layout: A) Multiple of 8, and B) Not a multiple of 8

See Labware on page 129 for acceptable labware at each deck location.

Transferring the samples to the microplate You can transfer the samples to the microplate that is supplied with the AssayMAP kits. See Starter kit, cartridges, and labware on page 128.

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

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2 If necessary, centrifuge the sample labware to remove bubbles.

Running the protocol

The Fractionation protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the peptides from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the 96AM Cartridge & Tip Seating Station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 131. Prepare the samples. See Preparing the samples on page 133. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs. Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide on page 574. Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge

Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 129.

About stacking Fraction Collection plates and Elution Buffer plates You must prepare stacks of pre-filled Elution Buffer plates and empty Fraction Collection plates on the Bravo deck before running the Fractionation protocol. You will use the Plate Stacking protocol in this app to stack the plates.

CAUTION You should always use the Plate Stacking protocol to stack the plates. Stacking the labware manually can produce misaligned stacks, resulting in crashes during the protocol run.

CAUTION Do not use labware that have been repeatedly heat sealed or subjected to extreme heat or cold. Warped labware can cause imprecise stacking, potentially causing head crashes during the protocol run.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Fractionation application.

To set up the Plate Stacking and Fractionation protocols: 1 Open the App Library. 2 Locate Fractionation, and then click App.

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The Fractionation application opens.

3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method.

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In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the protocol run on page 142. To modify the method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create or modify methods.

6 In the Application Settings area, specify the following to create or modify a method: a Select the labware you are using from the Labware (Locations 4 - 9) list. b In the Number of Fractions box, type a value from 0 to 6 for the fractions that

will be collected during the Fractionation protocol run. The value you specify must match the number Elution Buffer and collection plates you are using.

c Specify the cartridge settings:

Select the cartridge size from the list: 5 L Cartridges or 25 L Cartridges

In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 To save the method:

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a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 145.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 6) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

Equilibrate Aspirates Equilibration Buffer (deck location 9) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates samples (deck location 5) into the syringes, and then dispenses them through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 8).

5 L: 100 5 3

25 L: 100 5 3

Range: 0250 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through in the Flow Through Collection plate (deck location 8). If not selected, discards the sample flow-through in the Organic Waste plate (deck location 3).

Cup Wash Rinses the cartridge cups with Equilibration Buffer (deck location 9), and then discards the liquid into the Organic Waste plate (deck location 3).

5 L: 25 3

25 L:**

Range: 0100 010

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Internal Cartridge Wash

Aspirates Equilibration Buffer (deck location 9) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 8).

5 L: 50 10 3

25 L:**

Range: 0250 0.5500 010

Collect Flow Through

Collects the Internal Cartridge Wash flow- through at Flow Through Collection (deck location 8). If not selected, discards the Internal Cartridge Wash flow-through to Organic Waste (deck location 3).

Predispense Elution Buffer

Dispenses the specified volume of each Elution Buffer into its respective Fraction Collection plate before the start of each elution.

5 L: 15

25 L: 15

Range: 050

Elute Fraction 1

Aspirates Elution Buffer 1 (deck location 5 following its movement from deck location 4) into the syringes, and then dispenses it through the cartridges into the Fraction Collection Plate 1 (deck location 8 following its movement from deck location 7).

5 L: 25 5 1

25 L: 125 5 1

Range: 0-250 0.1-500 010

Elute Fraction 2

Aspirates Elution Buffer 2 (deck location 5 following its movement from deck location 4) into the syringes, and then dispenses it through the cartridges into the Fraction Collection Plate 2 (deck location 8 following its movement from deck location 7).

5 L: 25 5 1

25 L: 125 5 1

Range: 0-250 0.1-500 010

Elute Fraction 3

Aspirates Elution Buffer 3 (deck location 5 following its movement from deck location 4) into the syringes, and then dispenses it through the cartridges into the Fraction Collection Plate 3 (deck location 8 following its movement from deck location 7).

5 L: 25 5 1

25 L: 125 5 1

Range: 0-250 0.1-500 010

Elute Fraction 4

Aspirates Elution Buffer 4 (deck location 5 following its movement from deck location 4) into the syringes, and then dispenses it through the cartridges into the Fraction Collection Plate 4 (deck location 8 following its movement from deck location 7).

5 L: 25 5 1

25 L: 125 5 1

Range: 0-250 0.1-500 010

Elute Fraction 5

Aspirates Elution Buffer 5 (deck location 5 following its movement from deck location 4) into the syringes, and then dispenses it through the cartridges into the Fraction Collection Plate 5 (deck location 8 following its movement from deck location 7).

5 L: 25 5 1

25 L: 125 5 1

Range: 0-250 0.1-500 010

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the Plate Stacking protocol:

1 Click . The Plate Stacking protocol starts.

2 Follow the on-screen instructions to place the Elution Buffer plates at their specified locations. The protocol stacks the Elution Buffer plates at deck location 4.

3 Follow the on-screen instructions to place the Fraction Collection plates at their specified locations. The protocol stacks the Fraction Collection plates at deck location 7.

Elute Fraction 6

Aspirates Elution Buffer 6 (deck location 4) into the syringes, and then dispenses it through the cartridges into the Fraction Collection Plate 6 (deck location 7).

5 L: 25 5 1

25 L: 125 5 1

Range: 0-250 0.1-500 010

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 145. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 154. **The Cup Wash and Internal Cartridge Wash steps are not an option for the 25 L cartridges because of the volume limit of the wells in the labware at deck location 9. Instead, a cartridge wash should be done in place of one of the elution steps for the 25 L cartridges.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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To start the Fractionation protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol. The default protocol with 100-L samples and 5-L/min flow rate should take approximately 55 minutes to complete.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

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WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Fractionation protocol to

your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

Protocol stepwise guidelines

Protocol step Guidelines or notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply too

much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

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Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines or notes

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Prime This step removes entrained air from the packed resin bed and properly wets the surface of the resin. In preparation for priming, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (L). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the resin bed. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5300 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (C18 and RP-S), which are often used with the Fractionation application, require an equilibration solution that has a very low concentration or no organic solvent for effective binding during the sample loading step. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes and then mounts the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station.

IMPORTANT Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For details, see Preparing the sample plates on page 134.

This step is selected by default. Volume (L). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. Default: 100 Practical: 10250 Range: 0250 Note: The lower the sample volume, the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed into the Organic Waste plate. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.

Cup Wash This step removes the residual sample solution that may remain above the resin bed after the Load Samples step. The Cup Wash step aspirates Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Organic Waste plate, and then washing the syringes at the wash station. This step is selected by default for the 5 L cartridges. The Cup Wash and Internal Cartridge Wash steps are not an option for the 25 L cartridges because of the volume limit of the wells in the labware at deck location 9. Instead, a cartridge wash should be done in place of one of the elution steps for the 25 L cartridges. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash

This step uses Wash Buffer to wash non-specifically bound molecules from the resin bed. In preparation for the Internal Cartridge Wash, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Wash Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Wash Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. If the Load Samples step is selected, the first 5 L (5 L cartridges) of Wash Buffer is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash volume minus the chase volume is dispensed at the Internal Cartridge Wash flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. This step is selected by default for the 5 L cartridges. The Cup Wash and Internal Cartridge Wash steps are not an option for the 25 L cartridges because of the volume limit of the wells in the labware at deck location 9. Instead, a cartridge wash should be done in place of one of the elution steps for the 25 L cartridges. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except for syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min might not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the flow-through from Internal Cartridge Wash step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash is dispensed into the Organic Waste plate. This step is selected by default. This step is skipped if the Internal Cartridge Wash step is not conducted.

Predispense Elution Buffer

This step maximizes the recovery of small volume elutions (< ~ 20 L) by allowing direct elution into a liquid. Small volumes may cling to the end of the cartridge during elution that cannot be removed by the programmed tip touches in the wells of the plates. This step transfers a specified volume of Elution Buffer from the appropriate Elution Buffer plate (1-6) to the corresponding Fraction Collection plate before eluting each fraction into the Fraction Collection plate. Volume (L): Default: 15 Range: 050

Elute Fraction 16 Before running the Fractionation protocol, the Stack Plates protocol stacks a set of Elution Buffer plates at deck location 4 and a set of Fraction Collection plates at deck location 7. The number of Fraction Collections plates is equal to the Number of Fractions you specified. In preparation for elution, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During this step, the plate movements on the deck are as follows:

1 The Sample plate and Flow Through Collection plate move one position to the right and are stacked on the Priming Buffer plate and Equilibration Buffer plate, respectively.

2 To collect fractions 1-4, the Elution Buffer and Fraction Collection plates are moved from the stacks a locations 4 and 7 to deck locations 5 and 8 before each elution step.

3 The Elution Buffer and Fraction Collection plates for fractions 14 are stacked at deck locations 6 and 9 after each elution step. For example: Elution Buffer plate 1 is moved from the Elution Buffer plates stacked at location 4 to location 5. Fraction Collection plate 1 is moved from the Fraction Collection plates stacked at location 7 to location 8. Elution buffer is aspirated into the syringes from Elution Buffer Plate 1 at location 5. If the Predispense Elution Buffer step was selected, this volume will be dispensed to Fraction Collection plate 1 at location 8. Cartridges are mounted and samples are eluted from the cartridge directly into Fraction Collection plate 1. The cartridges are parked and the syringes are washed at the wash station. Fraction Collection plate 1 is moved from location 8 and stacked on location 9. Elution Buffer plate 1 is moved from location 5 and stacked on location 6.

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4 For fraction 5, step 2 is conducted but instead of stacking the Elution Buffer and Fraction Collection plates (step 3), they remain at locations 5 and 8, respectively.

5 For fraction 6, there is no plate movement. Elution Buffer plate 6 remains at location 4 and Fraction Collection plate 6 remains at location 7.

Final Fraction Collection plates 1-4 will be in deck location 9, Fraction Collection plate 5 will be in deck location 8 and Fraction Collection plate 6 will be in deck location 7. Volume (L). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges, or 5075 L for 25 L cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Volume for 5 L cartridges:

Default: 25 Practical: 1030 Range: 0250

Volume for 25 L cartridges: Default: 125 Practical: 50150 Range: 0250

Note: Setting the volume to zero skips all Elute tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500 Wash cycle. The number of syringe washes to perform at the wash station after an Elute step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines or notes

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Automation movements during the protocol

Stack Plates protocol This section describes the basic movements of the AssayMAP Bravo Platform during the Stack Plates protocol. Changing the Number of Fractions will alter the movements.

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 L of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

Protocol step Guidelines or notes

Protocol step Head moves to deck location...

Action

Starting protocol 4 Checks the labware height.

7 Checks the labware height.

Stack Elution Buffer plates

8 to 4 Moves the Elution Buffer plate from deck location 8 to 4.

5 to 4 9 to 4 6 to 4 3 to 4

Moves the remaining Elution Buffer plates, in the order listed, to deck location 4.

Stack Fraction Collection plates

8 to 7 Moves the Fraction Collection plate from deck location 8 to 7.

5 to 7 9 to 7 6 to 7 3 to 7

Moves the remaining Fraction Collection plates, in the order listed, to deck location 7.

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Fractionation protocol This section describes the basic movements of the AssayMAP Bravo Platform during the Fractionation protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Starting protocol 4 Checks the Elution Buffer plate stack height.

7 Checks the Fraction Collection plate stack height.

9 Checks the labware height.

6 Checks the labware height.

2 Parks all cartridges that might have been loaded on the head from a previously aborted protocol.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes.

Prime Cartridges 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Performs an external probe wash.

6 Aspirates 10 L of Priming Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates the Priming Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Priming Buffer through the cartridges and into Organic Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

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Equilibrate Cartridges 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Performs an external probe wash.

9 Aspirates 10 L of Equilibration Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Equilibration Buffer into the cartridge cups and exercises the cartridges off task.

9 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Equilibration Buffer through the cartridges into the Organic Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Load Samples 5 Aspirates the samples into the syringes.

2 Mounts the cartridges on the head.

8 Dispenses the samples through the cartridges and into the Flow through Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash (5 L cartridges only)

9 Aspirates the Wash Buffer.

2 Washes the cartridge cups and exercises the cartridges off task.

3 Dispenses the Wash Buffer into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Internal Cartridge Wash (5 L cartridges only)

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Performs an external probe wash.

9 Aspirates 10 L of Wash Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Wash Buffer into the cartridge cups and exercises the cartridges off task.

9 Aspirates the Wash Buffer.

2 Mounts cartridges on the head.

3 Dispenses 5 L Wash Buffer through the cartridges at the Load Samples flow rate for the sample chase step.

3 Dispenses the remaining Wash buffer through the cartridges at the Internal Cartridge Wash flow rate and into the Organic Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

5 Moves the Sample plate to deck location 6.

8 Moves the Flow Through Collection plate to deck location 9.

Protocol step Head moves to deck location...

Action

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Reference library

1 Russel, J., Van Den Heuvel, Z., Bovee, M. & Murphy, S., Workflow Automation for LC/ MS: In-Solution Protein Digestion, Peptide Cleanup, and Strong Cation-Exchange Fractionation of Peptides Enabled by AssayMAP Technology, Agilent Application Note 5991-3602EN, 2013

2 Russel, J., Van Den Heuvel, Z., Bovee, M., Murphy, S., Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-2957EN, 2016

See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Bravo Platform.

Elute Fractions 16 4 to 5 Moves Elution Buffer plate from deck location 4 to 5.

7 to 8 Moves Fraction Collection plate from deck location 7 to 8.

5 Aspirates the Elution Buffer for elution.

2 Mounts the cartridges on the head.

8 Elutes into the Fraction Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

8 to 9 Moves Fraction Collection plate from deck location 8 to 9.

5 to 6 Moves Elution Buffer plate from deck location 5 to 6.

Repeats for fractions 2, 3, and 4.

Repeats for fraction 5, except the final plates remain at deck locations 5 and 8.

Repeats for fraction 6, except the plates remain at deck locations 4 and 7. Note: Final Fraction Collection plates 1-4 will be at deck location 9. Fraction Collection plate 5 will be in deck location 8. Fraction Collection plate 6 will be at deck location 7.

Final Syringe Wash 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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This chapter contains the following topics: App description on page 160 Before you start on page 160 Preparing the solutions on page 165 Planning the cartridge layout on page 169 Running the protocol on page 171 Assay development guidelines and protocol notes on page 179 Reference library on page 192

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App description

IMAC Cartridge Customization v2.0. This application enables automated stripping and charging of AssayMAP Fe(III)-NTA cartridges with the desired metal of choice for immobilized metal affinity chromatography (IMAC) experiments. The protocol enables metal customization of from 1 to 96 cartridges in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the IMAC Cartridge Customization protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. You might need the following when preparing the reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

AssayMAP Fe(III)-NTA cartridges are available for performing phosphopeptide enrichment using the AssayMAP Bravo Platform. While Fe(III) is currently the most popular choice of metal cation for phosphopeptide enrichment, other metal cations such as Ga(III) and Zr(IV) have been proven effective for phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC) (ref 16). In addition, AssayMAP Fe(III)-NTA cartridges can be functionalized with metals such as Ni(II), Co(II), Zn(II), or Cu(II) to perform IMAC purification of histidine-tagged proteins or metal-binding proteins. The following table lists the available AssayMAP cartridges for performing IMAC Cartridge Customization on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Fe(III)-NTA cartridge rack G5496-60085

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or to fill remaining positions in columns that are partially occupied by Fe(III)-NTA cartridges, if necessary. For details, see Preparing the buffers and reagents on page 167.

G5496-60009

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Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges: Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

AssayMAP Fe(III)-NTA cartridges are stable from approximately pH 2 to 11. At levels approximately > pH 3.5, resin within the cartridges may turn from pale yellow to golden yellow to orange or brown due to the formation of iron(III) complexes (most commonly with hydroxide). Resin coloration and intensity are a function of both the pH and the chemical nature of the solutions passed through the cartridge. Bare NTA cartridges are stable from approximately pH 2 to 14.

AssayMAP Fe(III)-NTA cartridges that have been stripped (bare NTA cartridges) should be charged and used immediately.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

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Figure Labware locations on the Bravo deck (top view)

Reagents

The volume, type, and concentration of reagents required for cartridge customization vary depending on sample characteristics and the desired analytical result. Consult published literature (Reference library on page 192) for reagent recommendations for

Labware Mfr part number* Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 39

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 39

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 38

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 7, 9**

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 7, 9**

96 Greiner 650201 U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 4, 5, 6, 8, 9

Reservoir, Axygen Scientific RES-SW96-LP, 86mL, pyramid bottom

Axygen Scientific RES-SW96-LP

4, 5, 6, 8, 9

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms

Agilent 201254-100 4, 5, 6, 8, 9

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner and BioRad PCR plates are not compatible with the 25 L cartridges at deck location 7.

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sample and surface chemistry combinations. Also, see the Agilent AssayMAP Bravo Citation Index for published papers that use the IMAC Cartridge Customization application on the AssayMAP Bravo Platform. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 165.

Preparing the solutions

The following solutions are required for the IMAC Cartridge Customization protocol: Priming Buffer Metal Stripping Buffer Cartridge Wash Buffers Metal Reagent

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Metal-binding capacity

The metal-binding capacity of each AssayMAP NTA cartridge is >100 nmol of Fe(III) per cartridge as determined by inductively coupled plasmaoptical emission spectroscopy (ICP-OES).

Using the Reagent Volume Calculator for IMAC Cartridge Customization

The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, whether to perform the Collect Flow Through options, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns.

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Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figure shows the Reagent Volume Calculator.

Figure IMAC Cartridge Customization Calculator worksheet

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Removing macromolecular particulates

Make sure the solutions are free of macromolecular particulates, such as undissolved or precipitated salts. Use a 0.22-m filter to filter any salt-containing solutions and minimize the possibility of clogging the AssayMAP cartridges.

CAUTION A build-up of salts within the syringe barrels can corrode the syringe seals. Therefore, you should filter salt-containing buffers before use. In addition, you should use the System Startup/Shutdown v3.0 User Guide on page 574 utility to clean the syringes after every protocol run.

Preparing the buffers and reagents

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the protocol for stripping and charging Fe(III)-NTA cartridges.

Table Reagent preparation

Reagent (deck location)

Composition and comments

Priming Buffer (deck location 4)

To properly wet the surface of the resin and remove entrained air, use a priming solution containing a high amount of organic solvent. Organic solvent in the solution helps to purge entrained air within the cartridge resin bed and ensure the resin is properly wetted. The following buffer has been used successfully with the Fe(III)-NTA cartridges. 50% ACN : 50% H2O

Metal Stripping Buffer (deck location 5)

A chelator is necessary to remove the pre-charged iron from AssayMAP Fe(III)-NTA cartridges. Ethylenediaminetetraacetic acid is a strong chelator of metal cations and an aqueous solution is able to strip away metals bound to the NTA resin at a pH that is neutral to slightly basic. The following buffer has been used successfully to strip metal from the Fe(III)-NTA cartridges. 100 mM EDTA, pH 8.0

Cartridge Wash Buffer 1 (deck location 6)

This aqueous solution is used to flush away any remaining Metal Stripping Buffer and prepares the cartridge resin with a solution compatible with the Metal Reagent. The following buffer has been used successfully with the Fe(III)-NTA cartridges. 10 mM HCl, 50 mM acetic acid, or 0.1% TFA

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Note: All suggested solutions listed as percentages are volume/volume formulations.

Dispensing the solutions

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and subsequently be dispensed into the resin bed, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. See Using the Reagent Volume Calculator for IMAC Cartridge Customization on page 165. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

Metal Reagent (deck location 9)

This aqueous solution is used to immobilize metal cations to the surface of the bare NTA cartridge. Many aqueous metal solutions are highly acidic (pH < 1). Brief exposure of the resin bed to low-pH conditions during the Load Metal step will not harm the NTA resin within cartridges. The following reagent has been used successfully with the Fe(III)-NTA cartridges. 50150 mM metal salt in H2O

Cartridge Wash Buffer 2 (deck location 8)

This aqueous solution flushes any remaining Metal Reagent from the cartridges and leaves them in a state ready for immediate use or for short-term storage. For storage guidelines, see AssayMAP cartridges on page 162. Cartridge Wash Buffer 2 should not cause metal precipitation, form highly stable metal complexes with the immobilized metal, or contain a strong chelator that could strip the immobilized metal from cartridges. Consult relevant literature for specific metals to determine chemical compatibility of selected reagents. The following buffer has been used successfully to load Fe(III) on the Fe(III)-NTA cartridges that have been stripped of Fe(III) using this application. 10 mM HCl, 50 mM acetic acid, or 0.1% TFA

Reagent (deck location)

Composition and comments

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IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the cartridge positions in the 96AM Cartridge & Tip Seating Station. See Planning the cartridge layout on page 169.

To dispense the solutions into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Priming Buffer into the plate or reservoir to be placed

at deck location 4. 3 Add the specified volume of Metal Stripping Buffer into the plate or reservoir to be

placed at deck location 5. 4 Add the specified volume of Cartridge Wash Buffer 1 into the plate or reservoir to

be placed at deck location 6. 5 Add the specified volume of Cartridge Wash Buffer 2 into the plate or reservoir to

be placed at deck location 8. 6 Add the specified volume of Metal Reagent into the plate or reservoir to be placed

at deck location 9. 7 If necessary, centrifuge the buffer and reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518. See Labware on page 163 for acceptable labware at each deck location.

Planning the cartridge layout

Before transferring the reagents, you should plan the layout of the cartridges in the microplate. Consider the following: You can process 1 to 96 AssayMAP Fe(III)-NTA cartridges in parallel. The position

of the cartridges in the seating station dictates the position of the reagent and buffer solutions in the microplates and reservoirs.

If you have fewer than 96 Fe(III)-NTA cartridges, make sure the cartridges occupy full columns in the seating station, as the figure below shows. The default protocol settings assume that cartridges will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

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If the number of Fe(III)-NTA cartridges you have is not a multiple of 8, use AssayMAP Resin-Free cartridges to fill the empty well positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash steps.

Figure Examples of cartridge and reservoir layout: A) Multiple of 8 Fe(III)-NTA cartridges, B) Fe(III)-NTA cartridges and Resin-Free cartridges

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Running the protocol

The IMAC Cartridge Customization protocol: Washes the syringes. Primes the cartridges. Strips the iron from the cartridges. Removes the Metal Striping Buffer from the cartridges. Charges the cartridges with the desired metal of choice. Removes unbound metal from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the cartridge seating station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the buffers and reagents. See Preparing the solutions on page 165.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v3.0 User Guide.

Transfer the cartridges to the 96AM Cartridge & Tip Seating Station. See Cartridge Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. See Cartridge use and storage guidelines on page 163.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the IMAC Cartridge Customization application.

To set up the protocol: 1 Open the App library. 2 Locate IMAC Cartridge Customization, and then click App.

The IMAC Cartridge Customization application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 177.

To modify the method or create a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

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9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 179.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 4) into the syringes, and then dispenses it through the cartridges into the Waste plate (deck location 3).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

Strip Metal Aspirates Metal Stripping Buffer (deck location 5) into the syringes, and then dispenses it through the cartridges into Flow Through Collection (deck location 7), or into the Waste plate (deck location 3).

5 L: 50 5 3

25 L: 250 5 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Strip Metal flow- through at Flow Through Collection (deck location 7). If not selected, discards the flow- through into the Waste plate (deck location 3).

Cup Wash 1 Rinses the cartridge cups with Cartridge Wash Buffer 1 (deck location 6), and then discards the liquid into the Waste plate (deck location 3).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash 1

Aspirates Cartridge Wash Buffer 1 (deck location 6) into the syringes, and then dispenses it through the cartridges into Flow Through Collection (deck location 7), or into the Waste plate (deck location 3).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles.

Collect Flow Through

If selected, collects the Internal Cartridge Wash 1 flow-through at Flow Through Collection (deck location 7). If not selected, discards the Internal Cartridge Wash 1 flow- through into the Waste plate (deck location 3).

Load Metal Reagent

Aspirates Metal Reagent (deck location 9) into the syringes, and then dispenses through the cartridges into Flow Through Collection (deck location 7), or into the Waste plate (deck location 3).

5 L: 100 5 3

25 L: 100 5 3

Range: 0250 0.1500 010

Collect Flow Through

If selected, collects the flow-through from Load Metal Reagent at Flow Through Collection (deck location 7). If not selected, discards the Load Metal Reagent flow-through into the Waste plate (deck location 3).

Cup Wash 2 Rinses the cartridge cups with Cartridge Wash Buffer 2 (deck location 8), and then discards the liquid into the Waste plate (deck location 3).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash 2

Aspirates Cartridge Wash Buffer 2 (deck location 8) into the syringes, and then dispenses it through the cartridges into Flow Through Collection (deck location 7), or into the Waste plate (deck location 3).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash 2 flow-through at Flow Through Collection (deck location 7). If not selected, discards the Internal Cartridge Wash 2 flow- through into the Waste plate (deck location 3).

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see the Protocol stepwise guidelines on page 180. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 188.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner and BioRad PCR plates are not compatible with the 25 L cartridges at deck location 7.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

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After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol. The default protocol should take approximately 60 minutes to complete.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

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a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the IMAC Cartridge

Customization protocol to your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

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Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply

too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

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Prime This step removes entrained air from the resin bed and properly wets the surface of the resin. In preparation for priming, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Metal Waste plate, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Metal Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. For the most effective priming of AssayMAP Fe(III)-NTA cartridges, the Priming Buffer requires that the solution contain at least 50% organic solvent. Higher concentrations of organic solvent are also acceptable. This step is selected by default. Volume (L). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the cartridge. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Strip Metal This step removes the metal coordinated to the Fe(III)-NTA resin by using a strong chelator, such as EDTA. Complete removal of the bound metal from the resin bed is critical so that a different metal can be bound at the maximum metal-binding capacity of the cartridge. In preparation for stripping, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Metal Waste plate, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Strip Metal step aspirates the specified volume of Metal Stripping Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through cartridges into the Metal Waste plate or into the Flow Through Collection plate. The exterior of the cartridge tips are washed at the wash station to remove any residual buffer on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete removal of bound metal using a sufficiently concentrated chelator in the Metal Stripping Buffer. The default volume is probably much higher than required to strip the metal from the cartridges, but lower volumes have not been tested. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Strip Metal step tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default will likely have no benefit, but will increase the total assay time. A flow rate > 15 L/min using the default volume might not permit full penetration into the pores of the resin across the full length of the cartridge bed leaving residual metal on the cartridge. Default: 5 Practical: 215 Range: 0.5500 Wash cycle.The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Protocol step Guidelines and notes

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Collect Flow Through

If this step is selected, the flow-through from the Strip Metal step is dispensed directly into the Flow Through Collection plate. If this step is not selected, the flow-through is dispensed directly into the Metal Waste plate. This step is not selected by default.

Cup Wash 1 This step removes the small volume of residual liquid that might remain above the resin bed after the Strip Metal step. The Cup Wash step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Metal Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Internal Cartridge Wash 1

This step uses Cartridge Wash Buffer 1 to wash any remaining Metal Stripping Buffer from the resin bed and equilibrate the resin bed with a buffer compatible with the Metal Reagent to be used in the Load Metal step. In preparation for Internal Cartridge Wash 1, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Metal Waste plate, 10 L of Cartridge Wash Buffer 1 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 1 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or the Metal Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Protocol step Guidelines and notes

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Collect Flow Through

If this step is selected, the flow-through from the Internal Cartridge Wash 1 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed into the Metal Waste plate. This step is not selected by default. For the maximum practical working volumes per well for specific labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Load Metal Reagent This step allows the target metal to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the metal loading begins. The assumption is that there is still liquid in the cups from the wash step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates Metal Reagent into the syringes, and then performs an external syringe wash at the wash station to remove any Metal Reagent remaining on the outside of the probes before mounting the cartridges. The Metal Reagent is dispensed through the cartridges into the Flow Through Collection plate or the Metal Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any Metal Reagent on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). The volume of the Metal Reagent should be balanced with the concentration of the Metal Reagent and the metal binding capacity of the cartridge to ensure a large molar excess of metal to metal binding sites in the resin bed. Default: 100 Practical: 50150 Range: 0250 Note: Setting the volume to zero skips all Load Metal Reagent tasks except syringe washing. Flow rate (L/min). A flow rate less than the default will likely have no benefit, but will increase the total assay time. A flow rate > 15 L/min using the default volume might not permit full penetration into the pores of the resin across the full length of the resin bed, leaving portions of uncharged NTA in the resin bed, which would reduce binding capacity. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Protocol step Guidelines and notes

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Collect Flow Through

If this step is selected, the flow-through from the Load Metal Reagent step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Metal Reagent step is dispensed into the Metal Waste plate. This step is not selected by default.

Cup Wash 2 This step removes the residual solution that may remain above the resin bed after the Load Metal Reagent step. The Cup Wash 2 step aspirates Cartridge Wash Buffer 2 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Metal Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (L). A volume less than the default might be insufficient for cup washing, while a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Internal Cartridge Wash 2

This step uses Cartridge Wash Buffer 2 to wash unbound metal from the resin bed. In preparation for Internal Cartridge Wash 2, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Metal Waste plate, 10 L of Cartridge Wash Buffer 2 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 2 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or the Metal Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to 0 skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min might not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the flow-through from the Internal Cartridge Wash 2 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from the Internal Cartridge Wash 2 step is dispensed directly into the Metal Waste plate. This step is not selected by default.

Protocol step Guidelines and notes

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Automation movements during the protocol

This section describes the basic automation movements of the AssayMAP Bravo Platform during the protocol using the default method. Changing the selections or parameters will alter the movements.

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the Final Syringe Wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Metal Waste plate. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, 10 L of Cartridge Wash 2 will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L of DI water into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide marginally improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

Protocol step Head moves to deck location...

Action

Starting protocol 2 Parks all cartridges that might have been loaded on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes for 3 cycles.

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Prime 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Metal Waste plate.

1 Washes the exterior of the syringe probes.

4 Aspirates 10 L of Priming Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

4 Aspirates the Priming Buffer.

2 Mounts the cartridges onto the head.

3 Dispenses the Priming Buffer through the cartridges into the Metal Waste plate to prime the cartridges.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Strip Metal 5 Aspirates the Metal Stripping Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Metal Stripping Buffer through the cartridges into the Metal Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 1 6 Aspirates the Cartridge Wash Buffer 1 into the syringes.

2 Washes the cartridge cups and exercises the cartridges off task.

3 Dispenses the Cartridge Wash buffer 1 into the Metal Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Internal Cartridge Wash 1

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Metal Waste plate.

1 Washes the exterior of the syringe probes.

6 Aspirates 10 L of Cartridge Wash Buffer 1 for the cartridge air- gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates the Cartridge Wash Buffer 1 into the syringes.

2 Mounts the cartridges on the head.

3 Dispenses the Cartridge Wash Buffer 1 through the cartridges into the Metal Waste plate.

1 Washes the exterior of the cartridge tips at the wash station.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Load Metal Reagent 9 Aspirates the Metal Reagent into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

3 Dispenses the Metal Reagent through the cartridges into the Metal Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 2 8 Aspirates the Cartridge Wash Buffer 2 into the syringes.

2 Washes the cartridge cups and exercises the cartridges off task.

3 Dispenses the Cartridge Wash buffer 2 into the Metal Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Internal Cartridge Wash 2

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Metal Waste plate.

1 Washes the exterior of the syringe probes.

8 Aspirates 10 L of Cartridge Wash Buffer 2 for the cartridge air- gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates Cartridge Wash Buffer 2 into the syringes.

2 Mounts the cartridges on the head.

3 Dispenses Cartridge Wash Buffer 2 through the cartridges into the Metal Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Final Syringe Wash 2 Moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Metal Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Reference library

1 Posewitz, M.C. & Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem., 1999, 71:2883-2892.

2 Seeley, E.H., Riggs, L.D. & Regnier, F.E. Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme. J. Chromatogr. B, 2005, 817:81-88.

3 Steen, H., Stensballe, A. & Jensen, O.N. Phosphopeptide purification by IMAC with Fe(III) and Ga(III). Cold Spring Harbor Protocols, 2007, 2007:pdb.prot4607.

4 Machida, M. et al. Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis. FEBS J., 2007, 274:1576-1587.

5 Feng, S. et al. Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis. Mol. Cell. Proteomics, 2007, 6:1656-1665.

6 Hsu, J.L. et al. Functional phosphoproteomic profiling of phosphorylation sites in membrane fractions of salt-stressed Arabidopsis thaliana. Proteome Sci., 2009, 7:42.

See the Agilent AssayMAP Bravo Citation Index for published papers that used the AssayMAP Bravo Platform.

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App description

Immobilization v3.0. This application enables the creation of custom affinity purification cartridges by automating the immobilization of antibodies and other affinity ligands, from 1 to 96 standard AssayMAP cartridges in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Immobilization protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. The following equipment is recommended when preparing the samples and reagents:

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Immobilization and Affinity Purification protocols on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges:

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Protein A (PA-W) cartridge rack G5496-60000 G5496-60018

AssayMAP Protein G (PG-W) cartridge rack G5496-60008

AssayMAP Streptavidin (SA-W) cartridge rack G5496-60010 G5496-60021

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of Protein A, Protein G, or Streptavidin 5- or 25-L cartridges are required. For details, see Preparing the samples on page 203.

G5496-60009

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Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

PA-W, SA-W, and PG-W cartridges tolerate brief exposure to pH as low as 2.0. The stability of the PA-W, SA-W, and PG-W cartridges after capturing additional affinity ligands should be determined empirically.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware Mfr part number* Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3, 5, 6, 7, 8

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Reagents

The volume, type, and concentration of reagents required to immobilize antibodies or other affinity ligands onto AssayMAP cartridges for subsequent affinity purification experiments will vary depending on sample characteristics and the desired analytical result. Consult the resources that cover general principles of affinity purification such as those listed in the Reference library on page 234. For examples of reagents used during the immobilization of affinity ligands to protein A, protein G, and Streptavidin, consult the published scientific literature including the publications that use the AssayMAP Bravo Platform listed in the Agilent AssayMAP Bravo Citation Index. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure due to the buildup of salts within the syringe barrels. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Immobilization Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 199.

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 3, 5, 6, 7, 8

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 38

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7, 9**

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 4, 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 3, 5, 6, 8

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner PCR plate is not compatible with the 25 L cartridges at deck location 7.

Labware Mfr part number* Deck location options

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Preparing the solutions

The following solutions are used for the Immobilization protocol: Priming & Equilibration Buffer Wash Buffers: Cartridge Wash 1, Cartridge Wash 2, and Stringent Syringe Wash Blocking Reagent

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Immobilization Reagent Volume Calculator

The Reagent Volume Calculator is a Microsoft Excel file that contains the following: Calculator worksheet. You enter the number of columns to process, whether to

perform the Collect Flow Through option, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

Reagent Recipe worksheet. You enter the concentrations of each component in your reagent, and the worksheet calculates the recipe volumes required.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change.

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Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figures show the worksheets of the Reagent Volume Calculator.

Figure Immobilization Reagent Volume Calculator worksheet

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Figure Immobilization Reagent Recipe worksheet

Preparing the buffers

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the AssayMAP immobilization chemistry.

Table Reagent preparation

Reagent (deck location) Composition and comments

Priming & Equilibration Buffer (deck location 3)

Typically a buffered aqueous solution with neutral pH and physiologic salt concentration, for example, Phosphate-Buffered Saline (PBS), similar in composition to the buffer solution used to prepare the sample. Note: The Immobilization application also uses this buffer to wash the cartridges during the re-equilibration step.

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Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the samples at deck location 4 and cartridge positions in the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 203.

To dispense the solutions into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Priming & Equilibration Buffer into the labware to be

placed at deck location 3. 3 Add the specified volume of Cartridge Wash Buffer 1 into the labware to be placed

at deck location 5. 4 If using two wash buffers, add the specified volume of Cartridge Wash Buffer 2 into

the labware to be placed at deck location 6.

Cartridge Wash Buffer 1 (deck location 5)

A high-stringency buffer (for example, a neutral buffer with high NaCl) or a low-stringency buffer (for example, PBS). The Immobilization application allows the use of one or two wash solutions. The buffer selection depends on a number of factors. First, consider whether a blocking agent will be used. If so, one would typically use the low stringency solution for Cartridge Wash Buffer 1 and a high stringency wash buffer for Cartridge Wash Buffer 2, which would remove the loosely bound affinity ligand and blocking reagent from the resin bed. If a blocking reagent is not used, you would typically use a high-stringency wash buffer, such as PBS with high salt for Cartridge Wash 1 and use a low-stringency wash for Cartridge Wash Buffer 2.

Cartridge Wash Buffer 2 (deck location 6)

A high-stringency buffer (for example, a neutral buffer with high NaCl) or a low-stringency buffer (for example, PBS). The composition of this buffer is often dictated by the composition of Cartridge Wash Buffer 1. Cartridge Wash Buffer 2 will be replaced by the Priming & Equilibration Buffer if the Re-Equilibrate step is selected.

Stringent Wash Buffer (deck location 8)

The composition can vary widely depending on the experiment. Typically, this buffer would match the composition of the elution buffer that will be used during the Affinity Purification run, which usually follows the Immobilization run.

Blocking Reagent (deck location 9)

This reagent is most commonly used with SA-W cartridges in which case a free biotin solution is used to bind to the unbound streptavidin molecules on the surface of the resin. This blocks the streptavidin molecules that are not bound to the affinity ligand from binding to molecules present in the sample loaded on the cartridges during a subsequent Affinity Purification run using these cartridges.

Reagent (deck location) Composition and comments

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5 Add the specified volume of Stringent Wash Buffer into the labware to be placed at deck location 8.

6 Add the specified volume of Blocking Reagent into the labware to be placed at deck location 9.

7 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To prevent evaporation, samples should be prepared immediately before running the protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to optimize the binding conditions (for example, pH). Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the sample composition

The optimal chemical environment for binding is generally similar for protein A, protein G, and streptavidin. Protein A and G resins bind selectively to antibodies.

Examine the scientific literature for differences in their affinity for antibody subtypes from different species.

Streptavidin resin binds selectively to biotinylated molecules. Protein A, protein G, and streptavidin are relatively unaffected by most sample

components, including those present in complex protein mixtures.

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What are optimal pH conditions? One of the most important considerations for optimizing binding conditions is the pH of the sample, which should be near neutral pH. Both low (2 to 3) and high (10 to 11) pH ranges can prevent binding to protein A or protein G resins. The sample should generally be: Protein A. Greater than pH 6. Protein G. Greater than pH 4.

What sample components cause concerns? Protein A, protein G, and streptavidin generally tolerate moderate levels of salt, non- ionic detergents, and mild denaturing reagents, such as urea, quite well. You should examine scientific literature for the known effects and tolerances of protein A, protein G, and streptavidin, keeping in mind that these tolerances may differ depending on the antibody species and subtype for protein A and protein G.

Does the antibody species and isotype in the sample match the cartridge binding specificity? Protein A and protein G bind a wide variety of antibody subtypes and species.1 Carefully consider the species and subtype of antibody when choosing between using an AssayMAP Protein A or Protein G cartridge for purification. The antibodies that bind to protein G largely overlap the set that binds to protein A. While protein A is the industry standard for purification and titer determination of human therapeutic antibodies, protein G is the standard for purification of antibodies used as bioanalytical tools, primarily because many antibody subtypes are generated in species that bind poorly to protein A, for example, mouse IgG1 and rat IgG1.2

Determining the volume of sample to load

The AssayMAP Immobilization protocol permits loading up to 1000 L of sample onto AssayMAP cartridges. For sample volumes > 250 L, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 L) of the Bravo 96AM Head.

What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity: Quantitative binding capacity. The maximum mass of the target molecule that can

bind to the cartridge in a single pass, where less than 10% of the load appears in the flow-through. This value is dependent on the sample load flow rate.

Total binding capacity. The maximum mass of the target molecule that can bind to the cartridge. This can be achieved only by loading significantly more of the target molecule than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity.

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for Protein A, Protein G, and Streptavidin cartridges.

What is the concentration of the affinity ligand in the sample? If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

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How much affinity ligand should I immobilize? The amount of affinity ligand to immobilize depends on the amount of target. Use at least a 5-fold molar excess compared to the amount of target to be captured. A low molar excess of affinity ligand (approximately 5-fold) requires a very slow loading flow rate (approximately 2 L/min). As the molar excess increases so too can the loading flow rate. The exact molar excess and the flow rate should be determined empirical as they depend on many factors such as the strength of the affinity interaction and the diffusion rate of the target molecule.

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows. The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use AssayMAP Resin-Free cartridges to fill the empty well positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash steps.

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Figure Example of sample microplate and reservoir layout: A) Multiple of 8 samples, and B) Not a multiple of 8

See Labware on page 197 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The reagent volume calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

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Running the protocol

The Immobilization protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. If applicable (for example, using SA-W cartridges), blocks free binding sites. Removes non-specific binding molecules from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the Cartridge & Tip Seating Station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 199. Prepare the samples. See Preparing the samples on page 203.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v3.0 User Guide.

Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the cartridge bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent Technologies does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 196.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Immobilization application.

To set up the protocol: 1 If not already open, open the App Library. 2 Locate Immobilization, and then click App.

The Immobilization application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 214.

To modify the method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

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9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 216.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

Equilibrate Aspirates Equilibration Buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates samples (deck location 4) into the syringes, and then dispenses them through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 100 5 3

25 L: 100 5 3

Range: 01000 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the sample flow-through to waste in the wash station (deck location 1).

Cup Wash 1 Rinses the cartridge cups with Cartridge Wash Buffer 1 (deck location 5), and then discards the liquid into the wash station (deck location 1).

5 L: 25 3

25 L: 25 3

Range: 0100 010

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Internal Cartridge Wash 1

Aspirates Cartridge Wash Buffer 1 (deck location 5) into the syringes, and then dispenses it through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash 1 flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the Internal Cartridge Wash 1 flow-through into the wash station (deck location 1).

Load Blocking Reagent

Aspirates Blocking Reagent (deck location 9) into the syringes, and then dispenses it through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 50 5 3

25 L: 250 5 3

Range: 0250 0.1500 010

Collect Flow Through

If selected, collects the Blocking Reagent flow- through in the Flow Through Collection plate (deck Location 7). If not selected, discards the Blocking Reagent flow-through into the wash station (deck location 1).

Cup Wash 2 Rinses the cartridge cups with Cartridge Wash Buffer 2 (deck location 6) and discards the liquid into the wash station (deck location 1).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash 2

Aspirates Cartridge Wash Buffer 2 (deck location 6) into the syringes, and then dispenses it through the cartridges into the Flow Through Collection plate (deck location 7) or into the wash station (deck location 1).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects Internal Cartridge Wash 2 flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards Internal Cartridge Wash 2 flow-through into the wash station (deck location 1).

Stringent Syringe Wash

Aspirates Syringe Wash Buffer (deck location 8) into the syringes, and then discards the liquid into the wash station (deck location 1).

5 L: 50 2

25 L: 50 2

Range: 0250 010

Re- Equilibrate

Aspirates Equilibration Buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 217. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 230.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner PCR plates are not compatible with the 25 L cartridges at deck location 7.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683.

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To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility.

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e Confirm that the labware and accessories on the AssayMAP Bravo deck match the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Immobilization protocol to

your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

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Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply

too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

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Prime This step removes entrained air from the packed resin bed and properly wets the surface of the resin. In preparation for priming, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP affinity purification cartridges (PA-W, SA-W, and PG-W) typically used with this application contain affinity ligands (proteins) covalently immobilized onto resin. These cartridge types should be primed with aqueous buffers containing no or low amounts of organic solvent or protein denaturants. Because the Equilibration Buffer is drawn from the same reservoir as the Priming Buffer, buffers that favor analyte binding with minimal non-specific binding should be used for both priming and equilibration. This step is selected by default. Volume (L). The default volume should be sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridges:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the resin bed. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate step tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes, and then performs an external syringe wash at the wash station to remove any sample remaining on the outside of the probes before mounting the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. The protocol accommodates sample volumes up to 1000 L to be dispensed through the cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L. Each syringe has a maximum capacity of 250 L. When sample volumes are greater than 250 L, the protocol will iteratively load samples onto cartridges. To determine the number and volume of the iterative load steps, the protocol uses the following formulas: # of times to load = total sample volume/250,

where # times to load is rounded up to nearest integer volume of each load = sample volume/# of times to load For example, if the total sample volume is 900 L, then: # times to load = 900/250 = 3.6, which is rounded up to 4 volume of each load = 900/4 = 225 If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

IMPORTANT Be sure to include the recommended labware-dependent volume overage to prevent air from entering the cartridge. For more information, see Preparing the sample plates on page 205.

To determine the volume of sample to load, see Determining the volume of sample to load on page 204. This step is selected by default.

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Volume (L): The volume of sample to load should be balanced with the sample concentration and the mass capacity of the cartridge. The lower the sample volume, the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Large sample volumes (> 250 L) may require slightly more excess sample due to evaporation. Default: 100 Practical: 101000 Range: 01000 Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than 5 L/min may not significantly increase analyte binding, but this is highly dependent on the molar ratio of the capture ligand compared to the target molecule. For examples of cases with flow rates of less than 5 L/min, see Agilent app notes 5991-9010EN and 5991-8445EN in the Reference library on page 234. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed in the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed directly into the wash station. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.

Protocol step Guidelines and notes

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Cup Wash 1 This step removes the residual sample liquid that may remain above the resin bed after the Load Samples step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 1

This step uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the resin bed. In preparation for Internal Cartridge Wash 1, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Cartridge Wash Buffer 1 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 1 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. If the Load Samples step is selected, the first 5 L (5 L cartridges) or 25 L (25 L cartridges) of Cartridge Wash Buffer 1 is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash volume minus the chase volume is dispensed at the Internal Cartridge Wash flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the flow-through from Internal Cartridge Wash 1 is dispensed in the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed at the wash station. This step is not selected by default.

Load Blocking Reagent

This step allows a reagent of defined composition to be flowed through the cartridge after ligand immobilization to help minimize non-specific binding in subsequent target purification steps. The Load Blocking Reagent step may be especially helpful when a sub-saturating amount of ligand is bound to the resin in the cartridge. In preparation for blocking, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Blocking Reagent is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Load Blocking Reagent step aspirates Blocking Reagent into the syringes, performs an external syringe wash at the wash station, mounts the cartridges, dispenses the Blocking Reagent through the cartridges at the specified flow rate to either the Flow Through Collection plate or the wash station. An external cartridge wash is performed at the wash station to remove any blocking reagent on the outside of the cartridge. The cartridges are parked at the seating station and the syringes are washed at the wash station. Select the Load Blocking Reagent step to minimize to non-specific binding during subsequent target purification steps. This step is selected by default.

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Load Blocking Reagent (continued)

Volume (L). The volume of Blocking Reagent to aspirate into the syringes and dispense through the cartridges. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Load Blocking Reagent tasks except syringe washing. Flow rate (L/min). A rate slower than the default will likely have no benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, which may result in incomplete blocking. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the liquid eluted during the Load Blocking Reagent step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Blocking Reagent step is dispensed into the wash station. Select this step if you want to collect the Blocking Reagent flow-through. This step is not selected by default.

Protocol step Guidelines and notes

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Cup Wash 2 This step removes the residual buffer that may remain above the resin bed after the Load Blocking Reagent step. This step aspirates Cartridge Wash Buffer 2 and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the previous step is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are removed at the seating station, and then the liquid in the syringes is dispensed into the wash station. The syringes are washed at the wash station. This step is selected by default. Volume (L). A volume less than the default may be insufficient for cup washing, while a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash 2 tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 2

This step uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules and Blocking Reagent from the resin bed. In preparation for Internal Cartridge Wash 2, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Cartridge Wash Buffer 2 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 2 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer from the previous step on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but will also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the liquid eluted during Internal Cartridge Wash 2 is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through is dispensed into the wash station. Select this step if you want to collect the flow-through generated during Internal Cartridge Wash 2. This step is not selected by default.

Stringent Syringe Wash

This step cleans the syringes with the Stringent Syringe Wash Buffer. The Stringent Syringe Wash step aspirates the Stringent Syringe Wash Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the wash station. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the run time. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

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Re-Equilibrate This step conditions the resin and immobilized ligand to prepare them for the next step in the workflow, for example, running the Affinity Purification protocol. In preparation for re-equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Re-Equilibration step aspirates the Equilibration Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. Do not allow cartridges to dry out. After re-equilibration, use immediately for affinity purification or store them short term in the receiver plate containing 200 L of Equilibration Buffer per well. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully re-equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Re-Equilibrate tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads in the cartridge resin bed. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Immobilization protocol using the default method. Changing the selections or parameters will alter the movements.

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 L of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. Note: In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

Protocol step Head moves to deck location...

Action

Starting protocol 2 Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes the specified number of times.

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Prime 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then washes the exterior of the syringe probes.

3 Aspirates 10 L of Priming & Equilibration Buffer for the cartridge air-gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

3 Aspirates the Priming Buffer.

2 Mounts the cartridges on the head.

1 Dispenses the Priming & Equilibration Buffer through the cartridges into the wash station, and then washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then washes the exterior of the syringe probes.

3 Aspirates 10 L of Priming & Equilibration Buffer for the cartridge air-gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

3 Aspirates the Priming & Equilibration Buffer.

2 Mounts the cartridges on the head.

1 Dispenses the buffer through the cartridges to the wash station.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Protocol step Head moves to deck location...

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Load Samples 4 Aspirates samples into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

7 Dispenses sample through the cartridges to load sample. Collects flow-through in the Flow Through Collection plate.

1 Washes the cartridge exteriors at the wash station.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 1 5 Aspirates Cartridge Wash Buffer 1 into the syringes.

2 Performs the cup wash and exercise the cartridges off task.

1 Dispenses buffer into the wash station.

1 Washes the syringes.

Internal Cartridge Wash 1

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys.

5 Aspirates 10 L of Cartridge Wash Buffer for the cartridge air-gap prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

5 Aspirates Cartridge Wash Buffer 1 into the syringes.

2 Mounts the cartridges on the head.

1 Dispenses 5 L (5 L cartridges) or 25 L (25 L cartridges) Cartridge Wash Buffer 1 through the cartridges at the Load Samples flow rate for the sample chase step.

1 Dispenses the remaining Cartridge Wash Buffer 1 through the cartridges at the Internal Cartridge Wash 1 flow rate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes at the wash station.

Protocol step Head moves to deck location...

Action

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Load Blocking Reagent

9 Aspirates Blocking Reagent into syringes.

1 Washes exterior of probes at the wash station.

2 Mounts the cartridges on the head.

1 Dispenses Blocking Reagent through the cartridges.

1 Washes the cartridge exteriors at the wash station.

2 Parks the cartridges in the seating station.

1 Washes the syringes at the wash station.

Cup Wash 2 6 Aspirates Cartridge Wash Buffer 2 into the syringes.

2 Performs the cup wash and exercise the cartridges off task.

1 Dispenses buffer into the wash station.

1 Washes the syringes at the wash station.

Internal Cartridge Wash 2

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then washes the exterior of the syringe probes.

6 Aspirates 10 L of Cartridge Wash Buffer 2 for the cartridge air-gap prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates Cartridge Wash Buffer 2 into the syringes.

2 Mounts the cartridges on the head.

1 Dispenses Cartridge Wash Buffer 2 through the cartridges.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes at the wash station.

Stringent Syringe Wash

8 Aspirates the Stringent Syringe Wash Buffer.

1 Dispenses the buffer into the wash station.

1 Washes the syringes at the wash station.

Protocol step Head moves to deck location...

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Reference library

1 Terry, M.P. in Handbook of Affinity Chromatography, Second Edition. (ed. D.S. Hage), 367-397 (CRC Press, 2005).

2 PROTEUS Protein G Antibody Purification Handbook, Mini & Midi spin columns, Bio- Rad Laboratories, Inc 2013.

3 Wu, S., Shen, M., Murphy, S. & Van Den Heuvel, Z., An Integrated Workflow for Intact and Subunits of Monoclonal Antibody Accurate Mass Measurements, Agilent Application Note 5991-8445EN, March 2018

4 Han, J., Van Den Heuvel & Murphy, S., A streamlined drug-to-antibody ratio determination workflow for intact and deglycosylated antibody-drug conjugates, Agilent Application Note 5991-9010EN, September 2019

See the Agilent AssayMAP Bravo Citation Index for published papers that used the AssayMAP Bravo Platform.

Re-Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then washes the exterior of the syringe probes.

3 Aspirates 10 L of Priming & Equilibration Buffer for the cartridge air-gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

3 Aspirate the Priming & Equilibration Buffer.

2 Mounts the cartridges.

1 Dispenses buffer through the cartridges into the wash station.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes at the wash station.

Final Syringe Wash 2 Moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys.

1 Washes the syringes at the wash station.

Protocol step Head moves to deck location...

Action

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8 In-Solution Digestion: Multi-Plate v2.0 User Guide

This chapter contains the following topics: App description on page 236 Before you start on page 236 Preparing the solutions on page 241 Preparing the samples on page 253 Running the Reagent Plate Setup protocol on page 255 Running the Digestion protocol on page 261 Assay development guidelines and protocol notes on page 268

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App description

In-Solution Digestion: Multi-Plate v2.0.This application enables automated digestion of 1 to 384 protein samples in up to four 96-well microplates in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP Starter Kit, labware, and reagents for running the In-Solution Digestion: Multi-Plate protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which is required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

Optional equipment. The following equipment is recommended when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 Peltier Thermal Station with STC controller

8 Custom plate nest

9 Thermal plate insert for efficient heat transfer on the Peltier Thermal Station

10 Orbital Shaking Station with Control Unit

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Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

Labware and starter kits

The In-Solution Digestion protocol works with a broad range of user-supplied reagents for denaturation, reduction, alkylation, and proteolysis steps. This protocol requires a specific set of labware that can be sourced from various vendors.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Labware The following table provides a complete list of labware options and the corresponding deck locations. The following figures show the nine Bravo deck locations for labware.

Figure Reagent Plate Setup labware locations on the Bravo deck (top view)

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

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Figure In-Solution Digestion labware locations on the Bravo deck (top view)

Starter kits Agilent offers starter kits for performing In-Solution Digestion and Peptide Cleanup, as these two applications are often performed sequentially. The following table lists the two starter kits that are available for In-Solution Digestion and Peptide Cleanup. Each starter kit contains both cartridges and labware.

Labware Manufacturer part number*

Deck location options

Reagent Plate Setup labware

96 Red PCR Insert + 96 Eppendorf 30129300, PCR, Full Skirt

Agilent insert (provided) and Eppendorf 30129300

4

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 5, 7

250-L pipette tips Agilent 19477-002 6

96 ABgene 1127, Deep Well, Square Well, Round Bottom

ABgene AB-1127 8

In-Solution Digestion labware

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 2, 5 9

96 ABgene 1127, Deep Well, Square Well, Round Bottom

ABgene AB-1127 3

96 Red PCR Insert + 96 Eppendorf 30129300, PCR, Full Skirt

Agilent insert (provided) and Eppendorf 30129300

4

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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For more information on the cartridges, see the AssayMAP cartridges on page 358 in the Peptide Cleanup 4.0 User Guide.

Starter kit labware

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

The following table lists labware that are included in the starter kits (G5496-60034 and G5496-60013). Note: Additional labware are included in the starter kits but are required for the Peptide Cleanup application only. For more information, see Peptide Cleanup v4.0 User Guide on page 355.

For details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Reagents

The volume, type, and concentration of reagents required to prepare for the In-Solution Digestion: Multi-Plate protocol depends on a combination of factors, including specific chemistry requirements, the number of samples to process, and volumes and concentrations of reagents necessary to conduct denaturation, reduction, alkylation, and proteolysis. The In-Solution Digestion Reagent Volume Calculator manages this complexity by preparing a reaction summary, optimized solution recipes, and microplate layouts for all master reagents based on your input values.

Kit Part number

AssayMAP Digestion and RP-S Cleanup Starter Kit Contains 96 reversed-phase (RP-S) cartridges and starter kit labware for In-Solution Digestion and Peptide Cleanup.

G5496-60034

AssayMAP Digestion and C18 Cleanup Starter Kit Contains 96 C18 cartridges and starter kit labware for In-Solution Digestion and Peptide Cleanup.

G5496-60013

Labware Part number

1.2 mL Deep-Well PolyPro Clear Plates (qty 2) ABgene AB-1127

96-Well U-Bottom PolyPro White Plates (qty 11) Greiner 650207

96-Well PCR Plates (qty 3) Eppendorf 30129300

12-Column Low-Profile Reservoirs (qty 4) Agilent 201280-100

96-Well Round-Bottom, Clear Plates (qty 2) Greiner 650201

250-L Pipette Tips Agilent 19477-002

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Consult published literature for reagent recommendations for sample and surface chemistry combinations. See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP In-Solution Digestion: Multi-Plate application. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 241.

Preparing the solutions

The In-Solution Digestion: Multi-Plate protocol accommodates a wide range of digestion chemistries so that previously optimized conditions can be easily transferred to this automated platform. Therefore, the reagents described here are categories of reagents used in the assay rather than specific recommendations.

Solutions required for the protocol

Table Required solutions

Solutions Number of plates required

Description

Syringe Wash Buffer

4 This is an optional solution used to solubilize and wash away protein that may have adsorbed onto the probes and syringes. The use of 50 mM NaOH has been found to be a suitable stringent syringe wash solution for several proteins. However, other protein-solubilizing solutions may be used depending on the specific protein sample inputs. Use the Single Liquid Addition utility (Single Liquid Addition v2.0 User Guide) to transfer 300 L per well of the wash solution to the four Greiner 96-Well Round Bottom, White Plates that will be stacked on deck location 2 by the Plate Stacking protocol before running the In-Solution Digestion protocol. During the protocol run, these plates serve two purposes. The plates are moved on top of the sample plates and serve as lids to protect the samples from light during the alkylation step of the protocol. The plates are also used as reservoirs for the syringe wash solution which is used to wash the syringes to prevent carry-over between sample loading steps.

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CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for In-Solution Digestion: Multi-Plate

The Reagent Volume Calculator is a Microsoft Excel file that contains the following: Reaction Setup worksheet. You to enter information about the samples, denaturant

and reductant, alkylant, dilution, and digestion. The calculator determines the concentrations and volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

Reagent Prep worksheet. This sheet displays the volumes and composition of the reagents required for the in-solution assay based on the input provided in the Reaction Setup worksheet. The Reaction Prep worksheet consists of four tables: Denaturation Mixture, Alkylant, Protease, and Diluent Mixture If you are using more denaturant than what is required, you can enter the larger mass in the Denaturation Mixer table. The Master Reagent requires iodoacetamide powder. If you are using more than what is required, you can enter the larger mass in the Alkylant table.

Master Reagents

1 The three master mix reagents (Denaturation mixture, alkylant, and protease) are placed in the reagent setup plate which serves as the source plate for distributing these reagents into the plates used during the In-Solution Digestion: Multi-Plate protocol. Use the Reagent Plate Setup protocol to dispense these three reagents to dedicated reagent plates. Fill an ABgene 1.2 mL Deep-Well Plate with the Master Reagent. See Setting up the Reagent Plate Setup protocol on page 255.

Diluent Mixture

1 This solution is used to dilute the sample following denaturation, reduction, alkylation and before enzyme addition. The solution typically contains deionized water, 1.0 M Tris at pH 8.1, and 0.5 M TCEP. This reagent is placed in an ABgene 1.2-ml deep-well plate which is placed at deck location 3 during the In-Solution Digestion protocol run. The volume per well required for the Diluent Mixture and the composition of the solution is determined using the Reagent Volume Calculator.

Solutions Number of plates required

Description

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Automated Plate Setup worksheet. This sheet displays the layout of the Master Reagent plate and the Diluent Mixture plate, including the volumes of the reagents that must be transferred into the designated wells when running the Reagent Plate Setup protocol.

Manual Plate Setup worksheet. This sheet displays the layout of the Denaturation Mixture plate, Alkylant plate, Protease plate, and Diluent Mixture plate, including the volumes of the reagents required for the plates used in the In-Solution Digestion protocol in case you prefer to manually pipette these reagents into the plates rather than use the Reagent Plate Setup protocol.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click Restore Defaults. 5 Modify the values in the green boxes as required to match your specific method. As

you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

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Reagent Volume Calculator Reaction Setup fields

The following tables describe the fields in the Reaction Setup worksheet.

Reaction Setup: Sample Input area Field Description Number of Samples

The number of samples you want to process. The number of samples should be a multiple of 8. Note: If the number of samples is not a multiple of 8, excess reagent consumption will occur. See Total Number of Sample Columns. Range: 8384 Default: 96

Total Number of Sample Columns

Read only. The calculator converts the Number of Samples to the Total Number of Sample Columns by rounding up to the nearest multiple of 8. Range: 148 Default: 12

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Number of Sample Plates

The number of labware containing the samples you want to process. To minimize the volume of reagent consumed, specify a greater number of sample plates where fewer wells are used in each plate. This requires fewer syringes during the liquid transfer, which minimizes reagent consumption. To minimize the number of labware used, specify fewer number of sample plates. For example, place eight columns of samples on one plate. Because more syringes are used during the liquid transfer, more reagents are consumed. To assess which scenario works best, use the Reagent Prep worksheet. Range: 14 Default: 2

Number of Columns per Sample Plate

Read only. The number of columns that occupy each sample plate. The value is calculated using the Total Number of Sample Columns and the Number of Sample Plates. Range: 112 Default: 6

Starting Column Number on Sample Plate

The column at which the samples begin in the sample plate. Note: The samples can begin in any column in the sample plate. Make sure the columns in the plates are contiguous. You cannot skip a column. If you have multiple sample plates, make sure the samples begins at the same column across all the plates. Range: 112 Default: 1

Sample Volume, L

The volume of the samples in each well.

IMPORTANT Make sure the labware you are using can accommodate the sample volume you specify, plus the additional volume that will be added during the course of the run. You can start with the default value, 15 L, because it satisfies a typical set of reaction parameters.

Range: 0270 Default: 15

Protein Concentration in Sample, g/ L

The concentration of the protein in the sample. The In-Solution Digestion protocol can accommodate a wide range of concentrations, which is largely dependent on downstream needs. Range: Varies Default: 15

Field Description

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Reaction Setup: Denaturant/Reduction area

Concentration of Denaturant in Sample, Molar

The concentration of urea or guanidine already present in the sample. Note: The acceptable range differs for each denaturant. In addition, the value you specify influences calculations for the Denaturation Mixture. Range: 08 for guanidine; 09 for urea Default: 0.0

Total Mass per Sample, g

Read only. The mass of the sample. The value is calculated using the Sample Volume and Protein Concentration. Range: Varies Default: 225

Field Description

Field Description Choose denaturant from dropdown

The denaturant you want to use. Select either Guanidine or Urea.

Denaturant Concentration for this step, Molar

The concentration of guanidine or urea that you want to use in the denaturation step. To skip denaturation, enter 0.

IMPORTANT If you are performing the Denaturation step, make sure the concentration is within the range for the denaturant. The permissible range ensures stability during the protocol run.

Range: 08 for guanidine; 09 for urea Default: 6.00

Denaturant Concentration in Master Reagent, Molar

Read only. The concentration of guanidine or urea in the Master Reagent. To adjust the calculated value, increase or decrease the Volume of Denaturation Mixture to Add. For example, increase the Volume of Denaturation Mixture to Add to decrease the Denaturant Concentration in Master Reagent. Range: 08 for guanidine; 09 for urea Default: 9.00

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Table Reaction Setup: Alkylation Reaction area

Reductant Concentration for this step, Molar

The concentration of the reductant for the Denaturation step. To skip reduction, enter 0. Range: 00.100 Default: 0.010

Amount of Reductant, total micromoles

Read only. The micromoles of reductant. The value is calculated using the Reductant Concentration and the Denaturation Mixture volume. Range: Varies Default: 0.450

Internal Standard Stock Concentration, pmol/L

Optional. The concentration of the internal standard stock solution you want to add to the Denaturation Mixture. Range: Varies Default: 0.0

Amount of Internal Standard in reaction, total picomoles

The total number of picomoles of the internal standard you want to include in the reaction. The value is converted to a final concentration at the indicated reaction steps (displayed in B. Reactant Concentrations Summary). Range: Varies Default: 0.0

Buffer Concentration for this step, Molar

The concentration of the pH buffer present during the denaturation step. Note: Values below 0.1 M have not been tested. Values above 0.35 M are out of range for the solutions used to prepare Master Reagents. Range: 0.1000.350 Default: 0.125

Volume of Denaturation Mixture to add, L

The volume of Denaturation Mixture that will be added into the sample. This mixture may include denaturant, reductant, internal standard, and pH buffer as defined by the experiment. Range: 0270 Default: 30

Field Description

Field Description Alkylant Concentration for this step, Molar

The concentration of alkylant desired at this step. Range: Varies Default: 0.020

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Reaction Setup: Dilution/Alkylant Quench area

Amount of Alkylant, total micromoles

Read only. The number of micromoles of the alkylant. The value is calculated from the concentration and volume added to the reaction, and the final concentration at the indicated steps is displayed in the B. Reactant Concentrations Summary table. Range: Varies Default: 1.020

Volume of Alkylant to add, L

The volume of alkylant to add. Using the default or minimum volume (5 uL) minimizes unnecessary dilution of the reaction and provides greatest flexibility in the volumes of other reactants. Range: 5250 Default: 6.0

Field Description

Field Description Denaturant Concentration for Digest step, Molar

The concentration of guanidine or urea in the digestion step. This value determines the volume of Diluent Mixture. If the sample does not contain any denaturant, enter 0. If the Denaturant Concentration is 0, the software will allow you to manually enter a volume in the Volume of Diluent Mixture if NO Denaturant field (the last row in the 4. Dilution/Alkylation Quench area) to control the buffering capacity in the digestion reaction. Range: Varies Default: 1.00

Reductant Concentration in the Diluent Mixture, Molar

The concentration of reductant necessary for quenching the alkylant. The final concentrations of reductant and alkylant at the indicated reaction steps are summarized in the B. Reactant Concentrations Summary table. Range: Varies Default: 0.0030

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Reaction Setup: Digestion area

Amount of Reductant added in this step, micromoles

Read only. The number of micromoles of reductant that is added during this step. The value is calculated using the Reductant Concentration and Volume of Diluent Mixture. Range: Varies Default: 0.630

Volume of Diluent Mixture to add, L

Read only. The volume of the Diluent Mixture you want to add. The value is calculated using many of the values you supplied in the table. Range: < 250 Default: 210.0

Volume of Diluent Mixture if NO denaturant, L

The volume of Diluent Mixture to add if Denaturant Concentration for Digest Step is 0. The Diluent Mixture contains additional reductant, if added, and pH buffer. This field appears only if you entered 0 for Denaturant Concentration for Digest Step (the first field in the 4. Dilution/Alkylation Quench area).

Field Description

Field Description Protease-to-Sample Ratio (mass:mass)

The protease-to-substrate protein (sample) ratio. The default value of 50 means the final digest reaction will contain 1 g of protease for every 50 g of protein in the sample. Typically, this ratio varies from 1:25 to 1:100. Refer to the recommendations of the protease supplier. Range: Varies Default: 50

Protease Master Reagent Concentration, g/L

The concentration of the Protease Master Reagent. This value and the Protease-to-Sample Ratio determine the Volume of Protease to Add. The Protease Master Reagent should be prepared from your protease stock solution. Range: 0.051.50 Default: 0.500

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Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

\

Use one of the following methods to set up the reagent plates: Automated Reagent Plate Setup Manual Reagent Plate Setup

To use Automated Reagent Plate Setup: The Master Reagent plate should be prepared at this stage.

1 In the Reagent Volume Calculator, display the Automated Plate Setup worksheet. See the following figure.

2 Add the designated volumes of Protease, Alkylant, and Denaturation Mixture into the assigned columns of the Master Reagent plate as shown in the A. Master Reagent Plate area of the worksheet.

3 Add the volumes of the Diluent Mixture into the assigned columns of the Diluent Mixture plate as shown in the B. Diluent Mixture Plate area of the worksheet.

Volume of Protease to Add, L

Read only. The volume of Protease to add. The value is calculated using the Protease-to- Sample Ratio and the Protease Master Reagent Concentration. You can compare this value with the Volume of Digest Reaction value, below. Its lower bound is the lowest recommended volume (5 uL), and the upper bound should be no more than 10% of the Volume of Digest Reaction, for best control of reaction pH. Range: 550 Default: 9.0

Buffer Concentration for Digest step, Molar

Read only. The final concentration of the buffer. The value is calculated using the starting concentration and volumes added from the fields above. Make sure the value remains within the range for optimal for pH control and protease activity. Range: 0.0500.080 Default: 0.060

Volume of Digest Reaction, L

Read only. The digest reaction volume. The value is calculated using the volumes shown above this field. Make sure the labware you are using can accommodate this volume. The labware should also accommodate any reagents you might want to add to this volume following the overnight digestion. Range: <= 300 Default: 270

Field Description

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Note: The Diluent Mixture plate is prepared manually because of the large volumes required.

4 To complete the setup, run the Reagent Plate Setup protocol. See Running the Reagent Plate Setup protocol on page 255.

Figure Example of Automated Plate Setup worksheet

To use Manual Reagent Plate Setup: 1 In the Reagent Volume Calculator, display the Manual Plate Setup worksheet. See the

following figure. 2 Use manual pipettes to prepare reagent plates for the Protease, Alkylant,

Denaturation, and Diluent reagents based on their respective plate layouts.

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Figure Example of Manual Plate Setup worksheet

Preparing plates for stacking

The In-Solution Digestion: Multi-Plate protocol requires a stack of five 96-well Greiner 650207 U-Bottom plates at deck location 2, regardless of the number of sample plates being used.

IMPORTANT The automated Bravo plate stacking is required to create a perfectly aligned stack, even if you do not run all the steps or if you have fewer than four sample plates.

To prepare the plates for stacking: 1 Label one plate to be used as a lid for the Protease plate, and label four plates to be

used as lids for the sample plates. The four plates that will be used for sample plate lids also function as Syringe Wash Buffer plates.

2 Fill each Syringe Wash Buffer plate with 300 L per well of buffer, such that the filled columns match the columns of samples in the sample plates. You may use manual pipettes for this task, or you may use the Reagent Transfer utility, Reagent Aliquot utility, or the Single Liquid Addition utility. Before using a utility, the system must be prepared using the System Startup Utility. For details, see the System Startup/Shutdown v3.0 User Guide on page 574.

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Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before run time or keep them covered until you run the protocol.

The In-Solution Digestion application can accommodate a wide range of protein concentrations, which is largely dependent on downstream needs. Viscous samples may require dilution. When preparing the samples, you must: Remove macromolecular particulates that might interfere with accurate pipetting. Determine the volume of samples. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris.

Adjusting the sample composition

The concentration of denaturant in the sample should be entered into the In-Solution Digestion Reagent Volume Calculator. See Using the Reagent Volume Calculator for In- Solution Digestion: Multi-Plate on page 242.

Determining the volume of sample to digest

The acceptable volume of the sample is highly dependent on the digestion conditions. Use the In-Solution Digestion Reagent Volume Calculator to help determine sample volume. See Using the Reagent Volume Calculator for In-Solution Digestion: Multi- Plate on page 242.

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 8 to 96 samples per 96 well plate in parallel, and up to four sample

plates. The samples should be arranged in contiguous columns, therefore, the samples should be in multiples of 8.

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The volume of samples is limited by the labware you use and the digestion reaction conditions. See Using the Reagent Volume Calculator for In-Solution Digestion: Multi-Plate on page 242. The total volume should also include any volume of liquids you intend to add post digestion.

Transferring the samples to the microplate You can transfer the samples to the microplate that is supplied with the AssayMAP Starter Kit. See Labware and starter kits on page 238.

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Single Liquid Addition utility to transfer the

samples. For instructions, see one of the following: If you have only one sample plate, see Reagent Transfer v3.0 User Guide on

page 525. If you have multiple sample plates, see Single Liquid Addition v2.0 User Guide

on page 542. 2 If necessary, centrifuge the sample labware to remove bubbles.

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Running the Reagent Plate Setup protocol

The Reagent Plate Setup protocol uses the AssayMAP Bravo Platform to transfer reagents in the Master Reagent plate to dedicated reagent plates. Note: If you have already manually prepared the reagents, you can skip this topic.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you If applicable, know which experiment ID to use to record the steps executed during

the utility and app runs. Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide.

Setting up the Reagent Plate Setup protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the application.

To set up the Reagent Plate Setup protocol: 1 Open the App Library. 2 Locate In-Solution Digestion: Multi-Plate, and then click App.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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The In-Solution Digestion: Multi-Plate application opens.

3 In the navigation pane on the right side of the form, click

. The Reagent Plate Setup form opens.

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4 If applicable, click Select Experiment ID.

The Experiments Editor opens.

5 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

6 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Running the Reagent Plate Setup protocol on page 259.

To modify or create a method, proceed to step 7. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

7 In the Application Settings area, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. For details, see Application Settings for Reagent Plate Setup on page 258. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experiment ID report is generated.

8 To save the method, click Save Method. In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Application Settings for Reagent Plate Setup

Setting or step

Description Value

Number of Sample Plates

Specifies the number of sample plates to be processed. Default: 1

Range: 14

Number of Columns per Plate

Specifies the number of columns in each reagent plate (Protease, Denaturation, and Alkylant) that will be filled with reagent aliquots. This number must be consistent with the number of columns of samples in each sample plate. The value is used with the Starting Column of Reagent Plates to determine which columns in the reagent plates will receive reagents.

Default: 1

Range: 112

Starting Column of Reagent Plates

Defines the first column in each of the reagent plates that will receive reagent aliquots. This value works with the Number of Columns per Plate to define the range of each reagent plate that will receive reagent.

Default: 1

Range: 112

Protease Storage Temperature

Specifies the temperature set-point that will be used for the Protease plate for the entire Reagent Plate Setup protocol. The temperature controller will not turn off after completion of the Reagent Plate Setup protocol. The assumption is that an In-Solution Digestion run will closely follow the Reagent Setup Run.

Default: 10 C

Range: 435 C

Columns of Tips in Pipette Tip Box

Specifies the number of full columns of 250 L pipette tips in the source tip box. The columns of pipette tips must be contiguous and contain 8 pipette tips per column. If specifying fewer than 12 columns, ensure that no pipette tips are present in the unspecified columns. Make sure that the empty columns are on the right side of the tip box.

Default: 12

Range: 112

Add Denaturation Mixture

Transfers the specified volume, plus the required overage, to the Denaturation Mixture plate.

Default: 30 L*

Range: 1250 L

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Running the Reagent Plate Setup protocol

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To run the Reagent Plate Setup: 1 Ensure that the reagent plates, pipette tips, and accessories are at the assigned

deck locations, as shown in the Deck Layout image of the form. At deck location 4, ensure that the Red PCR Plate Insert is installed with the Protease plate. Otherwise, the protease will not be transferred properly.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

Add Alkylant Transfers the specified volume, plus the required overage, to the Alkylant plate.

Default: 6 L*

Range: 1250 L

Add Protease Transfers the specified volume, plus the required overage, to the Protease plate.

Default: 9 L*

Range: 1250 L

* Input the value for the volume that you want to add to the samples during the digestion run. The Reagent Volume Calculator automatically adjusts the value to include the recommended overage and provides the volume that you need to add to the plate to transfer the desired volume. For example, if you set the Add Denaturation Mixture volume to 30 L, then 50 L will be added to each active well: 30 L for the samples and 20 L for overage.

Setting or step

Description Value

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WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Cleaning up after Reagent Plate Setup

When the protocol run is finished, make sure you:

1 Remove the seating station with the used tips (deck location 2), the Master Reagent plate (deck location 8), and the pipette tip box (deck location 6) from the Bravo deck.

2 Discard the excess Master Reagents and used pipette tips following appropriate waste disposal procedures.

3 Keep all remaining labware on the AssayMAP Bravo deck.

IMPORTANT Do not remove the Protease plate from deck location 4, or the stack of reagent plates from deck location 7. These reagents are in their appropriate positions for the In-Solution Digestion: Multi-Plate run.

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Running the Digestion protocol

The In-Solution Digestion: Multi-Plate default protocol does the following: Denaturant mixture is added to the samples. Samples are denatured and reduced at elevated temperature. The samples are alkylated. The samples are diluted with Dilution mixture. Protease is added to the samples.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the reagents, including the plates for stacking. See Preparing the

solutions on page 241. Prepare the samples. See Preparing the samples on page 253. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v3.0 User Guide.

IMPORTANT Before you run the In-Solution Digestion protocol, you must run the Plate Stacking protocol to stack of five 96-well Greiner 650207 U-Bottom plates at AssayMAP Bravo deck location 2. The Bravo plate stacking is a requirement even if you do not run all the steps or if you have fewer than four sample plates.

Stacking the wash plates and the lid for the protease plate

The In-Solution Digestion: Multi-Plate protocol requires a stack of five 96-well Greiner 650207 U-Bottom plates at deck location 2, regardless of the number of sample plates being used. Ensure that you have prepared the plates for stacking. For details, see Preparing plates for stacking on page 252.

To stack the prepared wash and lid plates: 1 Open the In-Solution Digestion: Multi-Plate app:

If the Reagent Plate Setup for In-Solution Digestion: Multi-Plate form is already open,

click in the navigation pane.

Otherwise, locate In-Solution Digestion: Multi-Plate in the App Library, and then click App.

The In-Solution Digestion: Multi-Plate application opens.

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2 Click in the navigation pane.

Follow the instructions that appear on the screen after the run starts.

Setting up the Digestion protocol

To set up the Digestion protocol: 1 Ensure that the In-Solution Digestion: Multi-Plate form is open.

2 If applicable, click Select Experiment ID.

The Experiments Editor opens.

3 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

4 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Digestion protocol run on page 266.

To modify or create a method, proceed to step 5. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

5 In the Application Settings area, do the following: a Specify the sample information and protease storage temperature. See

Application Settings for In-Solution Digestion protocol on page 264 for details. b Select the Conduct Step? check box of each step you want to perform, and then

specify the settings for the selected steps. For details, see, Application Settings for In-Solution Digestion protocol on page 264.

c Click .

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In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Application Settings for In-Solution Digestion protocol

The following tables give a brief description of each setting. For details, including the full and practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 268.

Table In-Solution Digestion sample and protease settings overview

Table In-Solution Digestion Step settings

Setting or Step* Description Value

Starting Sample Volume

The volume of sample in each well in the sample plate. Default: 15 L

Range: 0300 L

Number of Sample Plates

The number of sample plates on the AssayMAP Bravo deck. Default: 1

Range: 14

Protease Storage Temperature

The temperature set-point for the Protease plate (deck location 4) for the duration of the run. The temperature controller will turn off after completion of the In-Solution Digestion run. Note: The temperature of the wells will be slightly different than the Peltier set point.

Default: 10 C

Range: 437 C

Setting or Step*

Description Volume in L

Mix Cycles

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

Default: 3

Range: 110

Add Denaturation Mixture

Aspirates the Denaturation Mixture (deck location 7) into the syringes, and then dispenses it into Sample plate 1 (deck location 5). The solutions in Sample plate 1 are mixed based on the Mix Cycles value, and then the syringes are washed at the wash station (deck location 1) and in the Syringe Wash Buffer plate. This step repeats for each sample plate that is on the deck (locations 6, 8, and 9).

Default: 30 15 3

Range: 1250 030 010

Incubation (Denaturation, off-deck)

Pauses the run after the Add Denaturation Mixture step so that you can manually move the sample plates off deck for incubation, if required, for denaturation and/or reduction. After incubation, you manually place the sample plates back onto the Bravo deck and resume the protocol run.

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can decrease the volumes.

Add Alkylant Moves the Denaturation Mixture plate from deck location 7 to 3, aspirates the Alkylant (deck location 7) into the syringes, and then dispenses it into Sample plate 1 (deck location 5). The solutions in Sample plate 1 are mixed based on the Mix Cycles value, and then the syringes are washed at the wash station (deck location 1) and in the Syringe Wash Buffer plate. This step repeats for each sample plate that is on the deck (locations 6, 8, and 9).

Default: 6 15 3

Range: 1250 030 010

Incubation (Alkylation, on- deck)

Incubates the sample plates for the specified period (in minutes) on the deck.

Default: Time: 45 minutes

Range: 0180 minutes

Add Diluent Mixture

Moves the Denaturation Mixture plate from deck location 3 to 7, aspirates the Diluent Mixture (deck location 3) into the syringes, and then dispenses it into Sample plate 1 (deck location 5). The solutions in Sample plate 1 are mixed based on the Mix Cycles value, and then the syringes are washed at the wash station (deck location 1) and in the Syringe Wash Buffer plate. This step repeats for each sample plate that is on the deck (locations 6, 8, and 9).

Default: 210 15 3

Range: 1250 030 010

Add Protease Aspirates Protease (deck location 4) into the syringes, and then dispenses it into Sample plate 1 (deck location 5). The solutions in Sample plate 1 are mixed based on the Mix Cycles value, and then the syringes are washed at the wash station (deck location 1) and in the Syringe Wash Buffer plate. This step repeats for each sample plate that is on the deck (locations 6, 8, and 9).

Default: 9 15 3

Range: 1250 030 010

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

Default: 3

Range: 110

*For additional protocol guidelines, see Protocol stepwise guidelines on page 268. For a complete list of the robotic movements executed during a run, see Automation movements during Reagent Plate Setup protocol on page 269.

Setting or Step*

Description Volume in L

Mix Cycles

Wash Cycles

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Starting the Digestion protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

protoc

To start the Digestion protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form.

At deck location 7, ensure the Denaturation Mixture plate is stacked atop the Alkylant plate. Note: The Reagent Plate Setup protocol stacks the labware at deck location 7 automatically. If you prepared the reagent plates manually, you must stack the plates manually at deck location 7.

IMPORTANT Do not remove any of the reagent plates from the deck even if the reagent is not being used. Instead, you may use an empty microplate as a place holder. The protocol requires these labware to be in their defined positions to run properly. The protocol requires five plates for the stack at deck location 2 regardless of the number of sample plates.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run. To monitor the progress of the run, check the Status box.

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WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

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c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Digestion protocol to your

particular experimental design Automation movements during the protocols For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

Protocol stepwise guidelines

Protocol step Guidelines and notes

Initial Syringe Wash The Initial Syringe Wash step removes potential contaminants from the syringes. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. You can increase the number of wash cycles to better clean the syringes. However, be aware that increasing the number of wash cycles also increases protocol run time and reduced syringe lifespan.

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Automation movements during Reagent Plate Setup protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Reagent Plate Setup protocol.

Add Denaturation Mixture

The Add Denaturation Mixture step prepares the reaction for denaturation and may include denaturant, reductant, internal standard, and buffer based on user-specified experimental design. The Denaturation Mixture is made during the Reagent Plate Setup protocol as the components are automatically transferred from the Master Plate into the Denaturation Mixture plate.

Incubation (denaturation- off deck)

The denaturation incubation step is used when incubation for denaturation or reduction is required. The step pauses the protocol run so that you can manually move the sample plates off the Bravo deck, manually seal the plates using the PlateLoc Sealer or equivalent device, and incubate the plates for the desired length of time. After the incubation period, remove the seals from the plates, place the plates back on the Bravo deck, and then resume the protocol run.

Add Alkylant Depending on the specific reagent used for alkylation, the reaction might be light sensitive. To protect the alkylation reaction from light, the Bravo Platform covers the sample plates with wash plates for the duration of the alkylant incubation step.

Incubation (Alkylation on deck)

The Incubation for alkylation step incubates the lidded samples on the Bravo deck. At the end of the incubation period, the wash plates are removed from the sample plates.

Add Diluent Mixture The Diluent Mixture prepares the reaction for digestion by diluting the denaturant in the sample with buffer to a concentration in which the protease is active and optionally adding a reductant to quench unreacted alkylant.

Add Protease The In-Solution Digestion protocol is designed to accommodate any proteolytic enzyme. You can add more enzyme using the Single Liquid Addition utility. See Single Liquid Addition v2.0 User Guide on page 542.

Final Syringe Wash The Final Syringe Wash step removes potential contaminants from the syringes. During each Final Syringe Wash cycle, the head aspirates 250 L of DI water into the syringes using the bare probes, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions.

Protocol step Guidelines and notes

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Protocol step Head moves to deck location...

Action

Start protocol NA Sets the Peltier Thermal Station to the Protease Storage Temperature (C) specified in the form.

7 Checks the plate height.

1 Dispenses any liquid remaining in the syringes. Performs 3 up-and-down cycles of the syringes above the wash station to dry the syringes.

Prepares pipette tips 6 Mounts all the pipette tips present in the tip box. Note: The pipette tips are arranged in the tip box in full columns starting at column 1.

2 Parks the pipette tips in the seating station.

2 Mounts all the pipette tips in the seating station, except columns 13, with a head offset so that the pipette tips in column 4 are mounted onto column 1 (left most column) of the head.

6 Returns unwanted pipette tips to tip box.

2 Mounts the pipette tips in column 13 of the seating station and moves them to columns 1012 of the seating station.

Alkylant solution transfer

2 Mounts the pipette tips from seating station column 12 onto column 1 of the head.

8 Prewets the pipette tips with Alkylant solution.

8 Aspirates the Alkylant solution, including a small excess.

7 Dispenses the Alkylant solution.

8&7 Repeats the preceding two steps as necessary to complete filling the wells.

8 Dispenses the excess Alkylant solution back into the Alkylant solution.

2 Ejects the used pipette tips into column 1 of the seating station.

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Automation movements during Plate Stacking protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Plate Stacking protocol.

Protease solution transfer

5 Picks up the Denaturation Mixture Plate.

7 Puts the Denaturation Mixture Plate on the Alkylant Plate.

2 Mounts the pipette tips from column 11 of the seating station onto column 1 of the head.

8 Prewets the pipette tips with Protease solution.

8 Aspirates the Protease solution, including a small excess.

4 Dispenses the Protease solution.

8&4 Repeats the preceding two steps as necessary to complete filling the wells.

8 Dispenses the excess Protease solution back into the Protease solution.

2 Ejects the used pipette tips into column 2 of the seating station.

Denaturation Mixture transfer

2 Mounts the pipette tips from column 10 of the seating station onto column 1 of the head.

8 Prewets the pipette tips with Denaturation Mixture.

8 Aspirates the Denaturation Mixture, including a small excess.

7 Dispenses the Denaturation Mixture.

8&7 Repeats the preceding two steps as necessary to complete filling the wells.

8 Dispenses the excess Denaturation Mixture back into the Denaturation Mixture.

2 Ejects the used pipette tips into column 3 of the seating station.

Protocol step Head moves to deck location...

Action

Protocol step Head moves to deck location...

Action

Start protocol 2 Checks the stack height.

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Automation movements during Digestion protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Digestion protocol. Changing the selections or parameters will alter the movements.

Stack plates 9 Moves Syringe Wash plate 4 to deck location 2.

8 6 5 3

Moves Syringe Wash plates 3, 2, 1, and then the Protease Lid to deck location 2.

Protocol step Head moves to deck location...

Action

Protocol step Head moves to deck location...

Action

Start protocol Sets the Peltier Thermal Station to the temperature specified on the form.

2 Checks the stack height.

7 Checks the stack height.

2 Picks up the Protease Lid plate.

4 Puts the Protease Lid plate on the Protease plate.

Initial Syringe Wash 1 Washes the syringes

Add Denaturation Mixture

7 Aspirates the Denaturation Mixture.

5 Dispenses the Denaturation Mixture, and then mixes the sample.

1 Washes the syringes.

2 Picks up a Syringe Wash plate from the top of the stack.

5 Puts the Syringe Wash plate on top of the Sample plate, and then performs a Stringent Syringe Wash.

1 Washes the syringes.

For each remaining Sample plate: Repeats the Add Denaturation moves starting with aspirating Denaturation Mixture from deck location 7. Returns all Stringent Syringe Wash plates at deck locations 9, 8, 6, and 5 to the plate stack at deck location 2, preserving the plate order so that the same Syringe Wash plates are used for their respective Sample plates.

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Incubation Off deck Protocol pauses and incubation message appears. Operator must manually move Sample plates off deck for incubation, and then move them back on deck and click Continue to resume the run.

Add Alkylant 7 Picks up the Denaturation Mixture plate.

3 Puts the Denaturation Mixture plate on top of the Diluent plate.

1 Pauses to wait for temperature to reach set point +/- 5 C.

7 Aspirates the Alkylant.

5 Dispenses the Alkylant and mixes the sample.

1 Washes the syringes.

2 Picks up a Syringe Wash plate from the wash plate stack.

5 Puts the Syringe Wash plate on top of the Sample plate, and then performs a Stringent Syringe Wash.

1 Washes the syringes.

For each remaining Sample plate: Repeats the Add Alkylant moves starting with aspirating Alkylant from deck location 7.

3 Picks up the Denaturation Mixture plate.

7 Puts the Denaturation Mixture plate on top of the Alkylant plate.

Add Diluent Mixture 5 Picks up the Syringe Wash plate.

2 Puts the Syringe Wash plate on top of the stack.

3 Aspirates the Diluent Mixture.

5 Dispenses the Diluent Mixture and mixes the sample.

1 Washes the syringes.

2 Picks up a Syringe Wash plate from the top of the stack.

5 Puts the Syringe Wash plate on the Sample plate, and then performs a Stringent Syringe Wash.

1 Washes the syringes.

For each remaining Sample plate: Repeats the Add Diluent Mixture moves starting with picking up the Syringe Wash plate.

Protocol step Head moves to deck location...

Action

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Add Protease 4 Picks up the Protease Lid plate.

2 Puts the Protease Lid plate on top of the stack. Picks up Protease Lid plate.

3 Puts the Protease Lid plate on top of the Diluent plate.

5 Picks up the Syringe Wash plate.

2 Puts the Syringe Wash plate on top of the stack.

3 Aspirates the Protease.

5 Dispenses the Protease and mixes the sample.

1 Washes the syringes.

2 Picks up a Syringe Wash plate from the wash plate stack.

5 Puts the Syringe Wash plate on top of the Sample plate, and then performs a Stringent Syringe Wash.

1 Washes the syringes.

For each remaining Sample plate: Repeats the Add Protease moves, except the Protease aspiration happens before picking up of the Syringe Wash plate from the Sample plate and placing it on the stack at deck location 2. Returns all Syringe Wash plates at deck locations 9, 8, 6, and 5 to the plate stack at deck location 2, preserving the plate order.

3 Picks up the Protease Lid plate.

2 Puts the Protease Lid plate on top of the stack.

Final Syringe Wash 1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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App description

In-Solution Digestion: Single Plate v2.0. This application enables automated in-solution reactions of 1 to 96 samples in a single 96-well microplate format. The In-Solution Digestion: Single Plate application is designed to maximize simplicity and flexibility for low-to-medium throughput reactions. The procedure consists of five successive liquid- addition steps that each transfer a reagent or mixture of reagents into a common sample plate, followed by incubation on or off deck. The sample volume, incubation duration, incubation temperature, labware, and other parameters can be customized for each of the five addition steps, allowing for almost any manual in-solution reaction method to be automated on the AssayMAP Bravo Platform.

Primary features

The primary features are: Plate lidding. Enables sample and reagent plates to be introduced with microplate

lids to minimize light exposure and evaporation losses. On-deck incubation. Enables timed sample plate incubation after every reagent-

addition step at a user-defined temperature and for a user-defined duration. Mixed-mode pipetting. For each liquid-addition step, allows the user to choose bare

AssayMAP probes or 250-L pipette tips to transfer the liquid. Method saving. Enables creating methods for future use. A generic standard in-

solution protein digestion method is included that you can use for initial testing purposes or as a template for creating custom digestion processes.

Experiment ID database. Captures the steps executed and the settings used during each run of an application or utility.

Comparison to In-Solution Digestion: Multi-Plate

The following table lists the primary differences between the In-Solution Digestion: Single Plate application and the In-Solution Digestion: Multi-Plate application, which is also included in the Protein Sample Prep Workbench.

Feature In-Solution Digestion: Single Plate In-Solution Digestion: Multi-Plate

Throughput 8 to 96 samples per run (1 full plate) 8 to 384 samples per run (up to 4 full plates)

Digestion method Flexible You can define any digestion method (up to 5 liquid-addition steps per run).

Fixed Specifically designed to run the following: Denature/Reduce > Alkylate > Dilute > Digest.

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Before you start

This topic lists the required hardware, software, and labware for running the In-Solution Digestion protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which is required for running the AssayMAP protocols.

Figure AssayMAP Bravo Platform components

Labware options Variable You may select labware for sample and reagent plates from a pre-approved list.

Fixed You must use specific labware for every reagent and sample plate.

Pipetting options For each addition step, you may use pipette tips or the bare probes.

You may use only the bare probes.

Feature In-Solution Digestion: Single Plate In-Solution Digestion: Multi-Plate

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Optional equipment. The following equipment is recommended when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent Heated Incubator

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 Peltier Thermal Station with STC controller

8 Custom plate nest

9 Thermal plate insert for efficient heat transfer on the Peltier Thermal Station

10 Orbital Shaking Station with Control Unit

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

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Labware

The In-Solution Digestion: Single Plate application does not use AssayMAP cartridges. All pipetting operations are handled in 96-well microplates using either pipette tips mounted on the AssayMAP probes or the bare AssayMAP probes. Note: Pipette tips are required for addition steps that include a deep-well plate on the AssayMAP Bravo Platform. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware Manufacturer part number* Deck location options

250-L pipette tips (preloaded in 96AM Cartridge & Tip Seating Station, Agilent G5409-20025)

Agilent 19477-002 2

Lid, Universal** Agilent 200858-100 49

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 49

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 49

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 49

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 49

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 49

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 49

96 Costar 3363, PP Conical Bottom Corning Costar 3363 49

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Reagents

The types and amounts of reagents required to prepare for the In-Solution Digestion protocol depends on a combination of factors, including specific chemistry requirements, the number of samples to process, and volumes and concentrations of reagents necessary to conduct your digestion process. The Regent Volume Calculator for In-Solution Digestion calculates the reagent volumes required including the overages.

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 49

96 Eppendorf 96-500_V-bottom, Clear PolyPro Eppendorf 96/500 49

96 Eppendorf 96-1000_U-bottom, Clear PolyPro Eppendorf 96/1000 49

96 Waters 186005837, Clear PolyPro Waters 186005837 49

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 59

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 59

Reservoir, Seahorse 201254-100, PP, 1 Well, pyramid bottom

Agilent 201254-100 59

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96-LP

59

96 V11 Manual Fill Reservoir Agilent G5498B#049 59

96 Red PCR Insert + Eppendorf 30129300, PCR, Full Skirt

Agilent insert (provided) and Eppendorf 30129300

4

96 Red PCR Insert + Bio-Rad PCR, Hard-Shell, Low- Profile, Full Skirt

Agilent insert (provided) and Bio-Rad HSP-9611

4

96 Red PCR Insert + Greiner 652270, PCR, Full Skirt, PolyPro

Agilent insert (provided) and Greiner 652270

4

96 Greiner U-Bottom Thermal Insert + Greiner 650201_U-Bottom, Clear PolyPro

Agilent G5498B#126 and Greiner 650201

4

96 Greiner V-Bottom Thermal Insert + Greiner 651201_V-Bottom, Clear PolyPro

Agilent G5498B#126 and Greiner 651201

4

96 AbGene U-Bottom Thermal Insert + AbGene 1127, Square Well, Round Bottom

Agilent G5498B#127 and ABgene AB-1127

4

*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **Lidded plates are allowed at these deck locations, not the lid by itself. If you plan to use a lidded plate, ensure that you use only the approved lid type.

Labware Manufacturer part number* Deck location options

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All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. For more information, see Preparing the solutions on page 281.

Preparing the solutions

The In-Solution Digestion: Single Plate application accommodates a wide range of reaction chemistries so that previously optimized conditions can be easily transferred to this automated platform. Agilent recommends that you attempt to transfer your manual in-solution digestion reagent chemistries directly onto the automated platform. When preparing bulk reagents for use in this application, the total volume required (volume to be pipetted plus overage) for a reagent depends on the labware type. Different types of labware have different well geometries, which results in variability in the amount of excess reagent volume (overage) required to ensure reliable pipetting.

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for In-Solution Digestion, Single Plate

The Reagent Volume Calculator is a Microsoft Excel file that contains a worksheet. You enter the number of columns to process, the volume for each step in the protocol, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click Restore Defaults.

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5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tool tip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figure shows the worksheet of the Reagent Volume Calculator.

Figure Reagent Volume Calculator worksheet

Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

When preparing to do a digestion run, it will often be necessary to aliquot bulk reagents into their individual reagent plates. You can aliquot the reagents using manual pipettes, or use the Reagent Aliquot utility to transfer the reagents automatically. For details, see Reagent Aliquot v2.0 User Guide on page 518.

IMPORTANT If you are using fewer than 96 wells, make sure you fill the labware to correspond with the sample layout in the sample plate and the pipette tip positions in the seating station. For more information, see Preparing the samples on page 283.

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Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Affinity Purification protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Choose a sample plate with sufficient capacity to hold the total reaction volume,

including the sample volume and the volume for all the completed addition steps in the run.

Transfer the samples to the microplate you want to use for the protocol run.

Planning the microplate setup

Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The samples should be arranged in

contiguous columns. The position of the samples in the microplate dictates the positions of the buffer solutions in the microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate. The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. If using pipette tips for transfer, the AssayMAP Bravo Platform applies differential pressure to seat pipette tips based on the number of full columns of pipette tips. To achieve proper pipette tip seating, entire columns must be used.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes or the pipette tips, compromising performance.The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the solution and the length of the run but the recommended overages are fine for most standard runs.

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To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

Running the protocol

The In-Solution Digestion: Single Plate protocol performs up to five successive liquid- addition steps. Each addition step transfers a reagent or mixture of reagents into a common sample plate, followed by incubation on or off deck. The sample volume, incubation duration, incubation temperature, labware, and other parameters can be customized for each of the five addition steps.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 281. Prepare the samples. See Preparing the samples on page 283. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs. Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide on page 574. Runs that require pipette tips. Prepare the seating station with the appropriate

number and configuration of pipette tips to match the samples to be processed. To prepare the pipette tips, run the Pipette Tip Transfer utility. For details, see Pipette Tip Transfer v2.0 User Guide on page 512.

Setting up the protocol

Before you start the protocol, make sure the appropriate selections and values are specified in the In-Solution Digestion application.

To set up the protocol: 1 Open the App Library. 2 Locate In-Solution Digestion: Single Plate, and then click App.

The In-Solution Digestion: Single Plate application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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Note: The software includes an example method file that you can use as a template to create your own methods. To run the selected method, go to Starting the protocol run on page 290. To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 Under Input Sample Settings, specify the settings to meet the requirements of your run. For details, see Input Sample Settings on page 287.

7 Under Input Addition Step Settings, select the check boxes of the steps that you want to perform, and enter the values and labware selections for the selected steps. For details, see Input Addition Step Settings on page 288 Note: To avoid potential confusion when a experiment ID report is generated, ensure the following for any unselected addition steps: Addition Volume, Mixing Cycles, Incubation Duration, and Number of Wash

Cycles boxes are blank. Pause After Addition and Use Tips check boxes are cleared. Labware Selection is set to No Labware. Plate Lidded is set to No Lid.

8 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it. A series of error checks are performed. If any errors are detected, a message displays a description of the problem. For details, see Error messages on page 299.

Input Sample Settings

The following table gives a brief description of each setting you specify about the Sample Plate at deck location 4.

Table Input Sample Settings overview

Setting Description Value range

Number of Full Columns of Samples

Specifies the number of full columns of samples in the sample plate at deck location 4.

112

Sample Plate Labware

Specifies the type of labware or labware plus thermal insert that will be placed at deck location 4, which contains the samples to be digested. Note: A thermal insert is critical for proper temperature regulation by the Peltier Thermal Station at this location. Note: The selected plate must be able to hold the initial volume and the volume that will be added to this plate over the course of the run. Note: If a deep-well plate is selected, pipette tips are required for the liquid transfers. For labware options, see Labware on page 279.

See Labware on page 279.

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Input Addition Step Settings

The following table gives a brief description of each setting you specify for each of the five reagent addition steps. For examples of how to vary the addition steps by changing the Incubation and Pause After Addition settings, see Assay development guidelines and protocol notes on page 292. The values that you use can vary dramatically from experiment to experiment. For an example of an approach that uses values like those in the supplied method, Standard Digestion 1.mth, see Agilent Application Note 5991-4872EN in the Reference library on page 308.

Table Input Addition Step Settings overview

Sample Plate Lidded

Specifies whether the sample plate has a lid. If you select Lid, ensure that the sample plate has a universal

microplate lid present at the start of the run. For details see Labware on page 279.

If you select No Lid, ensure the sample plate does not have a lid at the start of the run.

Lid, No Lid

Starting Sample Volume

Specifies the volume of sample that is initially present in the sample plate at the beginning of the run.

01000 L

Setting Description Value range

Setting Description Value range

Conduct Step Indicates whether to perform the corresponding addition step. Selected, Not selected

Addition Name Specifies a name for the addition step.

Addition Volume Specifies the volume of reagent that will be transferred from the reagent plate into the sample plate at deck location 4. Note: The Reagent Deck Location column shows the deck location of the reagent plate. Note: Accuracy and precision decrease with volumes less than 5 L.

01000 L

Mixing Cycles Specifies the number of mixing cycles that will be used to mix the samples and reagents after the reagent addition. Note: The number of mixes must be determined empirically. In general, if the total volume in the wells are greater than 200 L or the viscosity is high, the mix cycles must be greater than 10.

0100

Incubation Duration

Specifies the amount of time that the sample plate will be incubated (deck location 4) after the reagent is added and the mixing is completed. The reagent addition step will not start until the reading on the Peltier Thermal Station is within 5 C of the specified incubation temp. Note: The combination of setting the Incubation Duration to 0 and selecting Pause After Addition allows off-deck incubation for as long as desired. This may be required for high-temperature incubations where evaporation or condensation on the plate lid is a concern.

01000 min

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can decrease the volumes. If your In-Solution Digestion protocol requires pipette tips, you must use pipette tips for the mock run. For instructions on how to set up the pipette tips in the seating station, see Pipette Tip Transfer v2.0 User Guide on page 512.

Incubation Temperature

Specifies the temperature set point of the Peltier Thermal Station during the sample plate incubation (deck location 4).

OFF, 4110 C

Pause After Addition

Pauses the protocol after completing the tasks in the step and before starting the next addition step. See Assay development guidelines and protocol notes on page 292 for instructions on effectively using the Pause feature.

Selected, Not selected

Labware Selection

Specifies the labware type that will be present at the corresponding Reagent Deck Location for the step.

See Labware on page 279.

Plate Lidded Specifies whether the reagent plate (deck locations 59) will have a lid during the run. If you select Lid, ensure that the reagent plate associated with the step

has a universal microplate lid present at the start of the run. For details see Labware on page 279.

If you select No Lid, ensure the reagent plate associated with the step does not have a lid.

No Lid, Lid

Use Tips for Addition

Specifies whether pipette tips (deck location 2) will be used instead of the bare probes to transfer this reagent into the sample plate. To use bare probes, clear the check box. To use pipette tips, select the check box.

You must fill the seating station with the appropriate number and configuration of pipette tips to match the samples to be processed. To prepare the pipette tips, run the Pipette Tip Transfer utility (Pipette Tip Transfer v2.0 User Guide on page 512).

Selected, Not selected

Number of Wash Cycles

Specifies the number of wash cycles to be performed at the wash station after the reagent addition step has completed. If the Use Tips for Addition check box is selected, this step washes the

pipette tips. If the Use Tips for Addition check box is cleared, the protocol washes

the bare syringe probes.

0 - 10

Setting Description Value range

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Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form. Place the filled Sample Plate at deck location 4 and the filled reagent plates at

their respective deck locations. Ensure that all plates that require lids have been properly lidded, and no lids

are present on other labware. If pipette tips are required, ensure the pipette tips are in the seating station at

deck location 2. Make sure the labware are properly seated on the Bravo deck.

IMPORTANT If lids are specified in the method, ensure that you use the universal microplate lid. See Labware on page 279. Ensure that the lids are on the corresponding plates.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

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WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

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c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck

match the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Guidelines and examples on how to vary the protocol settings Automation movements during an example protocol run For a description of the settings, see Input Sample Settings on page 287 and Input Addition Step Settings on page 288.

Examples of how to vary Addition steps by changing Incubation and Pause settings

The following examples demonstrate different ways of defining the following settings to produce different AssayMAP Bravo behaviors: Incubation Duration (min) Incubation Temperature (C) Pause After Addition Note: The choice to use a lid or not is experiment dependent. Agilent recommends using a lid when doing incubations at elevated temperatures if evaporation is a concern.

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Example 1. Basic addition of a reagent to the samples This example shows the settings for a simple addition step that uses the bare AssayMAP probes to transfer 10 L from the Reagent plate at deck location 5 into the Sample plate at deck location 4. The settings are as follows: Incubation Duration 0 min Incubation Temperature OFF Pause After Addition not selected

Figure Example 1 settings

Table Example 1: AssayMAP Bravo actions during the protocol

When to use these settings: In general, when incubation and deck manipulation are not required, set these addition steps as follows: Incubation Duration (min) to 0 Incubation Temperature (C) to OFF However, if an Addition step with no incubation follows a step that includes incubation, make sure that you account for the ramp time to change the temperature. For details, see Additional incubation control considerations on page 297.

Example 2: Addition with on-deck incubation This example uses the Peltier Thermal Station to automate an incubation step. The addition step settings include on-deck incubation after the liquid transfer to the Sample plate. The settings are as follows: Incubation Duration 45 min Incubation Temperature 37 C Pause After Addition not selected

Action Deck location

a Uses the bare probes to aspirate 10 L from the Reagent plate into the syringes.

5

b Dispenses the 10 L into the Sample plate. 4

c Performs three mix cycles in the Sample plate to mix the contents.

4

d Performs three syringe washes at the wash station. 1

e Continues on to Addition Steps 2 to 5. varies

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Figure Example 2 settings

Table Example 2: AssayMAP Bravo actions during the protocol

When to use these settings: Set the Incubation Duration and Incubation Temperature values for all addition steps where the Sample plate incubation will be conducted automatically on the AssayMAP Bravo deck.

Example 3: Addition with a pause This example shows settings for an addition step that includes a pause after the liquid transfer to the Sample plate. The settings are as follows: Incubation Duration 0 min Incubation Temperature OFF Pause After Addition selected

Figure Example 3 settings

Action Deck location

a Sets the Peltier Thermal Station to 37 C. Note: The heater is activated before the liquid transfer because it takes about 5 minutes for the temperature to reach the set point.

4

b Uses the bare probes to aspirate 10 L from the Reagent plate into the syringes.

5

c Dispenses the 10 L into the Sample plate. 4

d Performs three mix cycles in the Sample plate to mix the contents.

4

e Starts the incubation timer when the Peltier Thermal Station reaches 37 C.

f Performs three syringe washes at the wash station. 1

g Remains idle for the remainder of the 45-minute incubation time.

h Continues on to Addition Steps 2 to 5. varies

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Table Example 3: AssayMAP Bravo actions during the protocol

When to use these settings: Select the Pause After Addition option to pause the run so that you can do the following: Replace used pipette tips with new pipette tips. Remove the Sample plate from the deck for off-deck incubation.

Example 4: Addition with on-deck incubation and a pause This example uses the Peltier Thermal Station to automate an incubation step. After the incubation, the run will pause to allow the operator to modify something on the deck. This settings are as follows: Incubation Duration 60 min Incubation Temperature 60 C Pause After Addition check box selected

Figure Example 4 settings

Table Example 4: AssayMAP Bravo actions during the protocol

Action Deck location

a Uses the bare probes to aspirate 10 L from the Reagent plate into the syringes.

5

b Dispenses the 10 L into the Sample plate. 4

c Performs three mix cycles in the Sample plate to mix the contents.

4

d Performs three syringe washes at the wash station. 1

e A Pause message appears on the screen and the AssayMAP Bravo Platform stops executing new tasks. The action remains paused until you click the Continue button that appears on the screen.

f Continues on to Addition Steps 2 to 5. varies

Action Deck location

a Sets the Peltier Thermal Station to 60 C. Note: The heater is activated before the liquid transfer because it takes about 5 minutes for the temperature to reach the set point.

4

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When to use these settings: Use these settings to incubate a plate.

Example 5: Addition with only Incubation Temperature Set and Pause Activated This example uses the Peltier Thermal Station to partially automate an incubation step. The on-deck incubation of the Sample plate is activated by setting the Incubation Temperature, but no Incubation Duration is specified. The Pause After Addition option is selected, which will cause the incubator to be active during the pause so that the operator can control the duration of the incubation. The settings are as follows: Incubation Duration 0 min Incubation Temperature 60 C Pause After Addition selected

Figure Example 5 settings

b Uses the bare probes to aspirate 10 L from the Reagent plate into the syringes.

5

c Dispenses the 10 L into the Sample plate. 4

d Performs three mix cycles in the Sample plate to mix the contents.

4

e Starts the incubation timer when the Peltier Thermal Station reaches 60 C.

f Performs three syringe washes at the wash station. 1

g Remains idle for the remainder of the 60-minute incubation time.

h A Pause message appears on the screen and the AssayMAP Bravo Platform stops executing new tasks. The action remains paused until you click the Continue button that appears on the screen. Note: The Peltier Thermal Station remains at temperature (60 C in this case) until the Pause message is cleared.

i Continues on to Addition Steps 2 to 5. varies

Action Deck location

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Table Example 5: AssayMAP Bravo actions during the protocol

When to use these settings: Use this combination of settings when it is necessary to apply a robust seal to the plate, but still use the on-deck incubator to heat or cool the samples. Note: In this case, the operator is responsible for timing the incubation step because the system does not keep track of the time that the plate was removed or returned to the deck.

Additional incubation control considerations

IMPORTANT The Peltier Thermal Station requires time to reach the temperature set point. Therefore, the device must be activated before incubation at the set temperature begins.

The time that it takes the Peltier Thermal Station to reach the set temperature is called ramp time. The temperature is in flux during the ramp time. The ramp time duration greatly depends on whether the Peltier Thermal Station is being used to actively control the temperature to the next set point or not. The following table provides approximate temperature ramp times for common incubation scenarios.

Action Deck location

a Sets the Peltier Thermal Station to 60 C. Note: The heater is activated before the liquid transfer because it takes about 5 minutes for the temperature to reach the set point.

4

b Uses the bare probes to aspirate 10 L from the Reagent plate into the syringes.

5

c Dispenses the 10 L into the Sample plate. 4

d Performs three mix cycles in the Sample plate to mix the contents.

4

e Performs three syringe washes at the wash station. 1

f A Pause message appears on the screen and the AssayMAP Bravo Platform stops executing new tasks. The action remains paused until you click the Continue button that appears on the screen. Note: The Peltier Thermal Station remains at temperature (60 C in this case) until the Pause message is cleared.

g Continues on to Addition Steps 2 to 5. varies

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Table Examples of approximate ramp time (min) to reach target temperature

The following examples illustrate why temperature ramp times are an important consideration when defining an In-Solution Digestion method.

Example 1: Addition with incubation followed by addition with heater turned off

Figure Example 1 settings

In this example: Addition 1 requires an incubation of 45 minutes at 60 C. Addition 2 does not require incubation, so the Incubation Temperature is set to

Off. Intuitively, it makes sense to turn off the incubator if it is not required, but this could have unintended consequences for Addition 2 and subsequent additions. After completing Addition 1, the Peltier Thermal Station will be turned off in preparation for Addition 2, but its temperature will still be very close to 60 C. According to the preceding table of example ramp times, the Peltier Thermal Station requires at least 30 minutes to return to ambient temperature (~25 C). The elevated temperature at deck location 4, where the Peltier Thermal Station is installed, could be problematic if later addition steps are sensitive to elevated temperature. In most cases, this problem can be mitigated by using the Peltier Thermal Station to actively cool the temperature back to ambient temperature, as described in Example 2.

Scenario Uses Peltier Thermal Station?

Start temperature (C)

Target temperature (C)

Ramp time (min), approximate

1 Yes 25 60 < 5

2 Yes 25 10 < 5

3 Yes 60 10 < 10

4 Yes 37 25 < 5

5 No 37 25 > 15

6 No 60 25 > 30

7 No 10 25 > 20

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Example 2: Addition with incubation followed by addition with heater at ambient temperature

Figure Example 2 settings

In this example: Addition 1 requires an incubation of 45 minutes at 60 C. Addition 2 requires at 25 C (ambient temperature) at deck location 4. These settings will cause the Peltier Thermal Station to actively cool the temperature from Addition 1 to ambient temperature for Addition 2. The approximate time required is 5 minutes for this temperature change. See the preceding table of example ramp times.

Error messages

Before executing an In-Solution Digestion: Single Plate method, the application performs several error checks to ensure that a feasible run has been specified. The following table lists some of the types of error checks.

Error check Description

Sample Plate Capacity

Compares the Initial Sample Volume input to the Sample Plate Labware selection to verify that it does not exceed the labware volume capacity.

Reagent Plate Capacity

Compares each Reagent Addition Volume input to its respective Labware Selection to ensure that it does not exceed the labware volume capacity.

Cumulative Volume

Calculates the cumulative digest volume (Initial Sample Volume + sum of Reagent Addition Volumes) to ensure that the final digest volume does not exceed the Sample Plate Labware volume capacity.

Sample Plate Pipette Tip Compatibility

If a deep-well plate type is selected for the Sample Plate Labware, each addition step is verified to ensure that the Use Pipette Tips for Addition? option is selected.

Reagent Plate Pipette Tip Compatibility

If a deep-well plate type is selected for any of the five reagent Labware Selections, the corresponding Use Pipette Tips for Addition? setting is verified to ensure that pipettes will be used for the transfer.

No Addition Steps Checks to see that at least one reagent addition step has been specified by ensuring that not all of the Reagent Names settings have been set to NONE.

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If a problem is identified, an error message displays. The following table lists some of the error messages, reasons for the error, and a description of how to correct the error.

Method Status error message

Reason for error How to correct the error

File chosen is not a valid Method file.

The method file selected is for a different application.

Click Select Method, and then select a valid method file.

File type is incorrect. Please choose a correct Method file.

The file selected is not a method file.

Click Select Method, and then select a valid method file.

Error: Total volume will exceed Sample Labware capacity (0 L). Please select a larger capacity Sample Labware.

No labware is selected for the Sample Plate.

Under Input Sample Settings, select the labware that you are using from the Sample Plate Labware list.

Error: Total volume will exceed Sample Labware capacity (n L). Please select a larger capacity Sample Labware.

The initial sample volume exceeds the capacity of the selected labware type.

Do one of the following: Select a labware type with a larger capacity from

the Sample Plate Labware list. Reduce the value in the Starting Sample Volume

box to a number that is within the capacity of the current labware selection.

For a list of maximum volumes by labware type, see Labware on page 279.

Error: Sample Plate is a type of deep well labware. Tips are needed in all steps.

Some of the addition steps specify the use of the AssayMAP bare probes, which cannot access the bottom of most deep-well plates. Pipette tips are required for addition steps that include a deep-well plate on the AssayMAP Bravo Platform.

Do one of the following: Select the Use Tips for Addition check box for

each Addition step to ensure that pipette tips are used for the transfers instead of the AssayMAP probes.

If you do not want to use pipette tips for the transfers, ensure that no deep-well plates are selected in the Sample Plate Labware list and the Labware Selection lists for the Addition steps.

Error: Check Step 1 for volume/labware/ tip combination.

The Volume input for one or more of the Addition Steps is greater than the volume capacity for the selected labware type.

Do one of the following for the corresponding Addition step: Select a labware type with a larger capacity from

the Labware Selection list. Reduce the value in the Addition Volume box to a

number that is within the capacity of the current labware selection.

For a list of maximum volumes by labware type, see Labware on page 279.

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Error: Check Step 1 for volume/labware/ tip combination. Error: Total volume will exceed Sample Labware capacity. Please select a larger capacity Sample Labware.

The cumulative volume in the sample plate, after all addition steps have completed, would be greater than the capacity of the selected Sample plate labware type.

Do one of the following: Select a labware type with a larger capacity from

the Sample Plate Labware list. Reduce the values in the Starting Sample Volume

box and/or the Addition Volume boxes to volumes that are within the capacity of the current labware selection.

For a list of maximum volumes by labware type, see Labware on page 279.

There are no reagent steps included on the form. Please double check and start the protocol again.

The Addition Name for all five addition steps is set to None, which causes all steps to be skipped.

For each addition step you want to include, select or type a name in the Addition Name box. Ensure that you have entered settings for at least one addition step in the Input Addition Step Settings area.

Error: Incubation temperature for Addition step 1 is out of range.

The Incubation Temperature (C) is set to a value that is outside of the allowable range (4 to 100 C).

If you want to specify an on-deck incubation, enter a value within the allowable range (4 to 100 C) in the Incubation Temperature (C) box. If you do not want to specify a temperature, select OFF.

IMPORTANT For guidelines on how to control the temperature changes between addition steps, see Additional incubation control considerations on page 297.

Error: No labware associated with Addition step 1.

The Labware Selection is set to No Labware for one or more addition steps.

For each addition step you want to include, select the corresponding labware from the Labware Selection list.

Error Check Step 1 for volume/labware/tip combination.

A deep-well plate is selected for one or more of the addition steps, but these steps are specified to use AssayMAP Probes, which cannot access the bottom of most deep-well plates.

Do one of the following: Select the Use Tips for Addition check box for

each Addition step to ensure that pipette tips are used for the transfers instead of the AssayMAP probes.

If you do not want to use pipette tips for the transfers, ensure that no deep-well plates are selected in the Labware Selection lists for the Addition steps.

Error: Addition step 1 labware is a reservoir, lids cannot be used.

The selected labware type cannot have a lid, but the Plate Lidded setting is set to Lid.

Do one of the following: Select No Lid in the Plate Lidded box for this

Addition step. Select a type of labware that can use a lid in the

Labware Selection list for this Addition.

Method Status error message

Reason for error How to correct the error

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Automation movements during the protocol

In-Solution Digestion: Single Plate is designed to mimic common manual in-solution digestion procedures for successively adding digestion reagents to samples and then incubating. In this application, a 96-well sample plate is placed at Bravo deck location 4, and up to five 96-well plates containing digestion reagents are placed at deck locations: 59. During the protocol, the AssayMAP Bravo Platform transfers each of the digestion reagents into the sample plate, one at a time, with optional heated incubation steps after each reagent addition. The automation movements are dependent on the choices selected in the method. The following figure shows an example of an In-Solution Digestion method and the following table lists the automation movements for this example.

Figure Example method settings

Table Automation movements for example method

Protocol step Head moves to deck location...

Action

Starting protocol 2 Parks all cartridges that might have been loaded on the head from a previously aborted protocol.

1 Dispenses any liquid remaining in the syringes into the wash station.

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Addition step 1 1 Washes the syringes.

1 Sets the Peltier Thermal Station to 60 C and pauses until the temperature reaches 55 C.

5 Aspirates air into the syringes while moving to location 5.

5 Prewets the syringes by cycling up and down 3 times. Goes to the top of the wells and dispenses air and then performs a tip touch. Aspirates 20 L of Denaturation Mixture.

4 Picks up the lid.

3 Drops off the lid.

4 Dispenses 20 L of Denaturation Mixture, and then cycles up and down 15 times to mix the solution. Goes to the top of the wells and dispenses air and then does a tip touch.

3 Picks up the lid.

4 Drops off the lid.

1 Dispenses any air remaining in the syringes, and then washes the syringes. Note: The protocol pauses until you click Continue in the Scheduler Paused window.

Protocol step Head moves to deck location...

Action

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Addition step 2 1 Sets the Peltier Thermal Station to 25 C and pauses until the temperature reaches 30 C.

6 Picks up the lid.

3 Drops off the lid.

6 Aspirates air into the syringes while moving to location 6.

6 Prewets the syringes by cycling up and down 3 times. Goes to the top of the wells and dispenses air and then performs a tip touch. Aspirates 5 L of Alkylant.

3 Picks up the lid.

6 Drops off the lid.

4 Picks up the lid.

3 Drops off the lid.

4 Dispenses 5 L of Denaturation Mixture, and then cycles up and down 15 times to mix the solution. Goes to the top of the wells and dispenses air and then does a tip touch.

3 Picks up the lid.

4 Drops off the lid.

1 Dispenses any air remaining in the syringes, and then washes the syringes.

1 Waits for 30-minute incubation.

Protocol step Head moves to deck location...

Action

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Addition step 3 1 Sets the Peltier Thermal Station to 25 C and pauses until the temperature reaches 2025 C.

7 Aspirates air into the syringes while moving to location 7.

7 Prewets the syringes by cycling up and down 3 times. Goes to the top of the wells and dispenses air and then performs a tip touch. Aspirates 140 L of Diluent Mixture.

4 Picks up the lid.

3 Drops off the lid.

4 Dispenses 140 L of Diluent Mixture, and then cycles up and down 15 times to mix the solution. Goes to the top of the wells and dispenses air and then does a tip touch.

3 Picks up the lid.

4 Drops off the lid.

1 Dispenses any air remaining in the syringes, and then washes the syringes.

1 Waits for 0-minute incubation.

Protocol step Head moves to deck location...

Action

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Addition step 4 1 Sets the Peltier Thermal Station to 37 C.

1 Moves the syringes up and down for 4 cycles with a wicking across the chimneys after every cycle.

2 Mounts the pipette tips on the head.

8 Aspirates air into the syringes while moving to location 8.

8 Prewets the pipette tips by cycling up and down 3 times. Goes to the top of the wells and dispenses air and then performs a tip touch. Aspirates 5 L of Protease.

4 Picks up the lid.

3 Drops off the lid.

4 Dispenses 5 L of Protease, and then cycles up and down 15 times to mix the solution. Goes to the top of the wells and dispenses air and then does a tip touch.

3 Picks up the lid.

4 Drops off the lid.

1 Dispenses any air remaining in the syringes, and then washes the pipette tips.

2 Parks the pipette tips in the seating station.

1 Waits for 180-minute incubation.

Protocol step Head moves to deck location...

Action

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Addition step 5 1 Sets the Peltier Thermal Station to 25 C.

9 Aspirates air into the syringes while moving to location 9.

9 Prewets the syringes by cycling up and down 3 times. Goes to the top of the wells and dispenses air and then performs a tip touch. Aspirates 20 L of Acidification Reagent.

4 Picks up the lid.

3 Drops off the lid.

4 Dispenses 20 L of Acidification Reagent, and then cycles up and down 15 times to mix the solution. Goes to the top of the wells and dispenses air and then does a tip touch.

3 Picks up the lid.

4 Drops off the lid.

1 Dispenses any air remaining in the syringes, and then washes the syringes.

1 Waits for 0-minute incubation.

Protocol step Head moves to deck location...

Action

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Reference library

1 Bovee, M., Russel, J., Murphy, S., Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-4872EN, 2016

See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Bravo Platform.

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This chapter contains the following topics: App description on page 310 Before you start on page 310 Preparing cartridges with immobilized substrate on page 320 Preparing the solutions on page 315 Running the protocol on page 323 Assay development guidelines and protocol notes on page 333 Reference library on page 353

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App description

On-Cartridge Reaction v2.0. This application enables the automated aspiration of a temperature-controlled reagent, for example, an enzyme solution, through prepared AssayMAP cartridges that contain immobilized target molecules. This allows the separation of reaction products, simplifying downstream sample preparation and analytical processes. The protocol enables processing of from 1 to 96 reactions in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the On-Cartridge Reaction v2.0 protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. The following equipment is recommended when preparing the samples and reagents:

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the most common AssayMAP cartridges for performing the On-Cartridge Reaction on the AssayMAP Bravo Platform. But any AssayMAP cartridge can be used with this application. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges:

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Protein A (PA-W) cartridge rack G5496-60000 G5496-60018

AssayMAP Protein G (PG-W) cartridge rack G5496-60008

AssayMAP Streptavidin (SA-W) cartridge rack G5496-60010 G5496-60021

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of Protein A, Protein G, or Streptavidin 5- or 25-L cartridges are required. For details, see Planning the cartridge layout.

G5496-60009

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Use only primed and equilibrated cartridges. Note: The On-Cartridge Reaction app uses cartridges that have been primed in another application. For example, you might run Affinity Purification to immobilize samples on the cartridges that you want to use for On-Cartridge Reaction.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out rapidly if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution. See How to store prepared cartridges on page 320 for more details.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once. If cartridge reuse is desired (for example, use of cartridges with immobilized customer-supplied affinity ligands), the customer is responsible for determining acceptable performance. In this case, ensure that you use particulate-free samples, equilibrate the cartridges using a solution compatible with the surface chemistry and resin matrix, and never allow the cartridges to dry out.

PA-W, SA-W, and PG-W cartridges tolerate brief exposure to pH as low as 2.0. The stability of the PA-W, SA-W, and PG-W cartridges after capturing additional affinity ligands should be determined empirically.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

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Labware Manufacturer part number*

Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 39

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 39

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms

Agilent 201254-100 39

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES- SW96-LP

39

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 39

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 39**

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 39

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 39**

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 39

96 V11 Manual Fill Reservoir Agilent G5498B#049 39

96 Red PCR Insert + Eppendorf 30129300, PCR, Full Skirt

Agilent insert (provided) and Eppendorf 30129300

4

96 Red PCR Insert + Bio-Rad PCR, Hard-Shell, Low- Profile, Full Skirt

Agilent insert (provided) and Bio-Rad HSP-9611

4**

96 Red PCR Insert + Greiner 652270, PCR, Full Skirt, PolyPro

Agilent insert (provided) and Greiner 652270

4**

96 Greiner U-Bottom Thermal Insert + Greiner 650201_U-Bottom, Clear PolyPro

Agilent G5498B#126 and Greiner 650201

4

96 Greiner V-Bottom Thermal Insert + Greiner 651201 V-Bottom, Clear PolyPro

Agilent G5498B#126 and Greiner 651201

4

96 AbGene U-Bottom Thermal Insert + AbGene 1127, Square Well, Round Bottom

Agilent G5498B#127 and ABgene AB-1127

4

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner and BioRad PCR plates are not compatible with the 25 L cartridges at deck locations 3, 4, 7, and 9.

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Reagents

The volume, type, and concentration of reagents required for On-Cartridge Reaction vary depending on sample characteristics and the desired analytical result. For Agilent application notes and peer-reviewed papers using the On-Cartridge Reaction application, see the Reference library on page 353 and the Agilent AssayMAP Bravo Citation Index, respectively. These resources provide detailed examples of reagents used successfully with this application in a variety of workflows. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details about this utility and the reagent recommendations for washing the syringes, see the Syringe Wash v3.0 User Guide. All labware requires volume overage for the protocol to execute properly. Use the On- Cartridge Reaction Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 315.

Preparing the solutions

The following solutions are required for the On-Cartridge Reaction protocol: Equilibration & Chase Buffer Reagent (for example, an enzymatic or chemical agent) Cartridge Wash Buffers Elution & Syringe Wash Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for On-Cartridge Reaction

The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, whether to perform the Collect Flow Through options, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns.

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Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figure shows the On-Cartridge Reaction Reagent Volume Calculator.

Figure On-Cartridge Reaction Reagent Volume Calculator

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Determining the Reagent volume for deck location 4

The Reagent Volume Calculator determines a recommended volume for the Reagent (deck location 4) based on the reaction volume plus a standard overage for the labware type. The values for both the reaction volume and the overage are influenced by multiple factors. Note: The labware-based overage in the Reagent Volume Calculator is a general recommendation that can changed. The overage value may be higher or lower than the default based on the length of the protocol run, when the labware is used during the run, and the volatility of the solution used in the labware. The Reagent (deck location 4) has additional factors to consider when determining the overage volume to use. The Reaction volume is the volume that will be moved over the cartridge during the Reaction step, which is defined by the reaction time input. This volume is drawn up through the cartridges from the wells in two stages of the reaction:

1 First stage. The Initial Draw volume is drawn up through the cartridges at a relatively rapid rate to quickly replace the volume in the resin bed with the reaction solution (for example, enzyme solution). Agilent recommends using the default value for the Initial Draw: 4 L (5 L cartridges) or 20 L (25 L cartridges). The default value is slightly less than one column volume, which should be sufficient for replacing the dead volume in the resin bed.

2 Second stage. The difference between the Reaction volume and the Initial Draw is aspirated over the resin bed at a flow rate that is required for this volume to be aspirated through the resin bed for the Duration input on the form.

The Reaction volume cannot be less than the Initial Draw volume, but it can be equal to it. How much larger the Reaction volume is compared to the Initial Draw is dependent on how much volume one wants to draw over the resin bed during the reaction. The properties of the reaction (for example, stability of the reacting agent, cost, molar ratio of reactant to substrate required, and so forth) will drive this decision. When choosing an overage volume, consider the following: Dead volume required to ensure that air is not aspirated into the cartridge Evaporation, which is dependent on the temperature of the reaction Length of the reaction and the volatility of the sample during the sample loading

step Enough liquid must remain in the wells to act as a heat conductor to the resin bed. At least 35 L of liquid is required in each well of a 96-well PCR plate at the end of the incubation time, as this is the minimum volume required to maintain contact with the bottom of the cartridges. This contact is required to conduct heat into the resin bed. A PCR plate is the most practical and common labware type at deck location 4 because of the low volumes of solution typically used at that location and the low dead volume requirements for a PCR plate.

Example using 5 L cartridges Note: See the app notes in the Reference library on page 353. In this example, a PCR plate is seated in the Red PCR Plate Insert at deck location 4. To conduct a 30-minute reaction at a resin bed temperature of approximately 37 C with a 6 L total aspiration volume, the temperature should be set to 45 C and 12 L of enzyme solution should be added per well before the run is started. A volume of 12 L is required in this example because: 6 L will be aspirated (4 L initial draw and 2 L drawn through the resin bed over

the 30 minute reaction).

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3 L is expected to evaporate. 3 L is required at the end of the run to ensure heat conductance occurs during the

entire run. For more details, see the Reaction Volume and Temperature steps in Assay development guidelines and protocol notes on page 333.

Preparing the buffers and reagents

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent Technologies recommends the buffers listed in the following table as a starting point for optimizing the AssayMAP On-Cartridge Reaction chemistry.

Table Reagent preparation

Reagent and deck location Composition and comments

Equilibration & Chase Buffer (deck location 3)

This reagent should provide optimal conditions for the reaction step, so it is typically the solvent used for the reaction reagent. For an enzymatic On-Cartridge Reaction run, this would typically be the enzyme reaction buffer. This buffer should not disrupt the affinity interaction between the cartridge resin and immobilized substrate.

Reagent (deck location 4)

Contains molecules that modify the immobilized substrate during the on- cartridge reaction. Enzymatic reaction, this solution would contain the enzyme. Chemical reaction, this solution would contain chemically active

molecules.

Cartridge Wash Buffer 1 (deck location 5)

High-stringency buffer (for example, a neutral buffer with high NaCl) or a low-stringency buffer (for example, a neutral, mass-spec-compatible buffer). The On-Cartridge Reaction protocol is designed to allow up to two sequential washes. The buffer selection depends on a number of factors, such as: Whether Wash Buffer 2 will be used, which is often dependent on

whether a stringent wash is desired in the protocol. If so, Wash Buffer 1 would be a stringent buffer, and Wash Buffer 2 would be a less stringent wash buffer, which would remove the stringent component of the buffer before elution.

If a stringent buffer is not used, typically, you would use Wash Buffer 1 only. The buffer would be a low-stringency wash buffer, for example, a neutral mass-spec-compatible buffer.

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When preparing these reagents, you must: Remove macromolecular particulates. Adjust the buffer composition to optimize the pH conditions. Determine the volume of solutions required using the Reagent Volume Calculator. The prepared reagents should be kept in a closed container before use to avoid evaporation.

Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware columns or wells that correspond with the cartridge positions on the Cartridge & Tip Seating Station. For more information, see Planning the cartridge layout on page 321.

To dispense the reagents into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Equilibration & Chase Buffer into the labware to be

placed at deck location 3. 3 Add the specified volume of Reagent into the labware to be placed at deck

location 4.

Cartridge Wash Buffer 2 (deck location 6)

Low-stringency buffer (for example, a neutral, mass-spec-compatible buffer). The composition of this buffer is often dictated by sensitivity of downstream steps to components of Cartridge Wash Buffer 2, as the void volume in the cartridges (~2 L) will contain the last wash solution used before the elution step and end up in the eluate unless the Eluate Discard option is selected in the elution step.

Elution & Syringe Wash Buffer (deck location 8)

Typically a low pH, mass-spec friendly solution, but this is highly dependent on the nature of the affinity interaction to be broken. A key consideration is if the sample will be neutralized following elution. If so, a weak acid that is easy to neutralize would be a good choice (for example, 12 mM HCl with 100 mM NaCl for the interaction between protein A and an antibody). However, this type of elution solution may be relatively inefficient in breaking the affinity interaction and, therefore, may require more volume for complete elution (for example, 46 column volumes for complete elution of antibodies off a protein A cartridge). If neutralization is not a key consideration or minimizing elution volume is a critical driver, a strong acid is a better choice (for example, 5% acetic acid) because complete elution can be achieved in as little as 23 column volumes.

Reagent and deck location Composition and comments

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4 Add the specified volume of Cartridge Wash Buffer 1 into the labware to be placed at deck location 5.

5 If using two wash buffers, add the specified volume of Cartridge Wash Buffer 2 into the labware to be placed at deck location 6.

6 Add the specified volume of Elution & Syringe Wash Buffer into the labware to be placed at deck location 8.

7 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can run the Reagent Aliquot utility or Reagent Transfer utility to transfer the buffers and reagents to different labware. For instructions, see the Reagent Aliquot v2.0 User Guide on page 518 or Reagent Transfer v3.0 User Guide on page 525.

Preparing cartridges with immobilized substrate

How to prepare the cartridges

The On-Cartridge Reaction app facilitates reactions between a substrate immobilized on a cartridge and a soluble reactant. The cartridges containing the immobilized substrate are prepared before the On-Cartridge Reaction run using any of the other AssayMAP applications, typically, Affinity Purification.

How to store prepared cartridges

IMPORTANT Do not allow cartridges with immobilized substrate to dry out. If the cartridges do dry out, do not use them.

To avoid cartridges drying out, store them immediately after the Affinity Purification run in an AssayMAP cartridge rack and receiver plate assembly, where each chimney of the receiver plate housing a cartridge with an immobilized substrate contains 200 L of buffer. Use a buffer that is known to be compatible with the cartridges immobilized substrate and resin.

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Figure AssayMAP cartridge rack lid (1), rack with cartridges (2), and receiver plate (3)

Using this storage method, the tip of the cartridge is submerged in a buffer and prevents the cartridge bed from drying out. Place the lid on the cartridge rack and receiver plate stack to prevent anything falling into the cartridges. For storage longer than a few hours, the assembly should be stored in a refrigerator (410 C).

Planning the cartridge layout

You can process 1 to 96 cartridges with immobilized substrate in parallel. The position of the cartridges in the seating station dictates the position of the reagent and buffer solutions in the microplates at each deck location. If you have fewer than 96 cartridges, make sure the cartridges occupy full columns

in the microplate, as the figure below shows. The default protocol settings assume that cartridges will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based on the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of cartridges you have is not a multiple of 8, use AssayMAP Resin- Free cartridges to fill the empty well positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash steps.

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Figure Examples of microplate and reservoir layout: A) Multiple of 8 samples, B) Not a multiple of 8 samples

The positions of the cartridges at deck location 2 dictate the positions of the solutions in deck locations 3, 4, 5, 6, and 8. See Labware on page 313 for the acceptable labware at each deck location.

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Running the protocol

The On-Cartridge Reaction protocol does the following: Washes the syringes. Equilibrates the cartridges. Loads the Reaction reagent onto the cartridges and incubates the reaction at the

temperature set point. Collects soluble reaction products. Removes non-target elements from the immobilized reaction product. Elutes the immobilized reaction product from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the Cartridge & Tip Seating Station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 315. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs. Run the Startup utility to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide. Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge

Transfer v2.0 User Guide on page 506. This application uses cartridges that have been prepared during a preceding AssayMAP application, typically, Affinity Purification.

IMPORTANT Do not allow cartridges with immobilized substrate to dry out. If the cartridges do dry out, do not use them.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the On-Cartridge Reaction application.

To set up the protocol: 1 Open the App Library. 2 Locate On-Cartridge Reaction v2.0, and then click App.

The On-Cartridge Reaction application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 330. To create or modify the method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

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9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the full and practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 333.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Equilibrate Aspirates the Equilibration Buffer (deck location 3) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1) or into Flow Through Collection (deck location 7).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Equilibrate flow- through at Flow Through Collection (deck location 7). If not selected, discards the equilibration flow-through at the wash station (deck location 1).

Reaction Aspirates the Reagent (deck location 4) through the cartridges in two steps (see note), followed by aspirating a chase volume. The Reaction flow-through and chase volume are collected at Flow Through Collection (deck location 7), unless Combine with Eluate is selected. The sum of the Reaction volume and the Reaction Chase volume must be less than 250 L.

5 L: 6 See note. 3

25 L: 30 3

Range: 0250 010

Note: The initial draw volume is aspirated at 10 L/min. Any additional volume (reaction volume minus the initial draw volume) is aspirated at a flow rate appropriate to satisfy the Duration setting.

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Tempera- ture

Specifies the set point temperature of the Peltier Thermal Station at deck location 4 during the Reaction step. The temperature in the cartridge will be less than this setting.

5 L: Temperature: 25 C

25 L: 25 C

Range: 4110 C

Duration Specifies the total length of time to aspirate the Reagent (deck location 4) through the cartridges.

Range: Time (m): 30 (0180)

Initial Draw

Specifies the initial draw volume, which is aspirated at 10 L/min. Note: Any additional volume (reaction volume minus the initial draw volume) is aspirated at a flow rate appropriate to satisfy the Duration setting.

5 L: 4 10

25 L: 20 10

Range: 0250

Reaction Chase

Aspirates the Chase Buffer (deck location 3) through the cartridges, to flush soluble reaction products into the syringes. This step occurs immediately after the aspiration of Reagent, combining the soluble reagent products and chase buffer within the syringes.

5 L: 15 5

25 L: 75 5

Range: 0250 0.1500

Combine with Eluate

If selected, collects the soluble reaction products at Eluate Collection (deck location 9). If not selected, collects the soluble reaction products at Flow Through Collection (deck location 7).

Cup Wash 1 Rinses the cartridge cups with the Cartridge Wash Buffer 1 (deck location 5), and then discards the liquid into the wash station (deck location 1).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash 1

Aspirates the Cartridge Wash Buffer 1 (deck location 5) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1) or Flow Through Collection (deck location 7).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash 1 flow-through at Flow Through Collection (deck location 7). If not selected, discards the Internal Cartridge Wash flow- through at the wash station (deck location 1).

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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Cup Wash 2 Rinses the cartridge cups with the Wash Buffer 2 (deck location 6) and discards the liquid into the wash station (deck location 1).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash 2

Aspirates the Cartridge Wash Buffer 2 (deck location 6) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1) or Flow Through Collection (deck location 7).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash 2 flow-through at Flow Through Collection (deck location 7). If not selected, discards the Internal Cartridge Wash 2 flow- through at the wash station (deck location 1).

Stringent Syringe Wash

Aspirates the Syringe Wash Buffer (deck location 8), and then discards the liquid into the wash station (deck location 1).

5 L: 50 2

25 L: 50 2

Range: 0250 010

Elute Aspirates the Elution Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into Eluate Collection (deck location 9).

5 L: 25 5 1

25 L: 125 5 1

Range: 0250 0.1500 010

Eluate Discard

If selected, a specified initial volume of Eluate will be dispensed through the cartridges, and then discarded at the wash station (deck location 1).

5 L: 0

25 L: 0

Range: 0250

Existing Collection Volume

Specifies the volume of liquid present in the Eluate Collection plate (deck location 9) at the beginning of the run.

5 L: 0

25 L: 0

Range: 01000

Final Syringe Wash

Conducts the specified number of internal syringe washes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 333. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 349.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner and BioRad PCR plates are not compatible with the 25 L cartridges at deck locations 3, 4, 7, and 9.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

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After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the emergency stop pendant.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

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Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

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Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the On-Cartridge Reaction

protocol to your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply too

much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 10 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

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Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for the reaction during the Reaction step. In the case of an enzymatic reaction, the equilibration buffer would be the optimized enzyme reaction buffer. Ensure that the optimal reaction buffer does not break the affinity interaction holding the immobilized substrate on the resin bed. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow rate. A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min using the default volume may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Collect Flow Through

This step is used for optimizing or troubleshooting a protocol to ensure that the equilibration solution is not eluting the immobilized substrate off the cartridge. If this step is selected, the dispenses the flow-through during the Equilibrate step into the Flow Through Collection plate for downstream processing or for analysis. If this step is not selected, the flow-through from the Equilibrate step is dispensed directly into the wash station. This step is not selected by default.

Reaction This step enables the reaction in the cartridge resin bed. No liquid is removed or added to the cartridge cups before the reaction begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Reaction step aspirates the Reagent through the cartridges in two steps, and then aspirates a chase volume:

1 Initial Aspiration. Aspirates the Initial Draw volume at a flow rate of 10 L/min. 2 Secondary Aspiration. Aspirates any additional volume at a flow rate appropriate to

satisfy the Duration setting. For example, if the volume is set at 6 L and the time was set for 30 minutes, the remaining 2 L of the 6 L volume would be aspirated at a flow rate of ~0.068 L/min (2 L / (30 min 0.4 min)).

3 Reaction Chase Aspiration. Aspirates the chase at the specified volume and flow rate.

The Reaction flow-through and Reaction Chase are collected in the syringes, and then dispensed into the Flow Through Collection plate, unless Combine with Eluate is selected. In which case, the Reaction flow-through and the Reaction Chase are dispensed into the Elution plate. This step is selected by default.

Protocol step Guidelines and notes

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Reaction (continued)

Volume (L). The volume must be determined empirically. For an enzymatic reaction, the volume would depend on the enzyme. If the enzyme is robust enough to undergo the number of enzymatic cycles required to push the reaction to completion, the Initial Draw volume may be sufficient. If the enzyme undergoes a limited number of cycles, the enzyme in the resin bed may need to be replenished during the course of the reaction, and significantly more than the Initial Draw volume may be required. Volume for 5 L cartridge:

Default: 6 Practical: 415 Range: 0250

Volume for 25 L cartridge: Default: 30 Practical: 2075 Range: 0250

Flow Rate. The initial draw volume is aspirated at 10 L/min. Any additional volume (reaction volume minus the initial draw volume) is aspirated at a flow rate appropriate to satisfy the Duration setting. Wash Cycles: The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Scenario using default settings for 5 L cartridges:

1 The Peltier Thermal Station heats until the set point temperature 2C is reached. 2 The Bravo 96AM Head picks up the cartridges from the Cartridge & Tip Seating

Station. 3 The Reagent is aspirated through the cartridges: 4 L at a flow rate of 10 L/min and

2 L at a flow rate of 0.068 L/min. (The Duration of both aspiration steps is 30 minutes.)

4 An external cartridge wash is conducted at the wash station. 5 15 L Chase Buffer is aspirated at 5 L/min. 6 The cartridges are ejected into the Cartridge & Tip Seating Station. 7 The syringe contents are dispensed into the Flow Through Collection plate. 8 Three internal syringe wash cycle are conducted.

Protocol step Guidelines and notes

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Temperature This setting specifies the set point temperature of the Peltier Thermal Station during the Reaction step. The optimal temperature is a function of the reaction being conducted. The actual temperature in the cartridge will be less than this setting. Heat loss occurs as the heat transfers across the thermal plate insert, a small air gap between the plate insert and the wells of the plate, the plastic of the plate, the solution in the well, and the cartridge resin bed. For example, a temperature setting of 45 C results in a cartridge bed resin temperature of approximately 37 C using the Red PCR Plate Insert and a PCR plate. A thermal plate insert is critical when running a reaction at a temperature other than room temperature. Without the insert, the air gap between the Thermal Station and the wells of the plate results in a greater temperature differential between the setting in the application and the actual temperature in the cartridge bed. The temperature differential is not a constant percentage of the set point, so the preceding example should be considered only as a starting point for cartridge target temperatures other than 37 C in the PCR plates. In addition, plate types vary in heat transfer efficiency because of their differences in design, such as well shape.

IMPORTANT At least 35 L of liquid is required in each well of a 96-well PCR plate at the end of the incubation time as this is the minimum volume required to maintain contact with the bottom of the cartridges. This contact is required to conduct heat into the resin bed. Longer incubation times or higher temperature settings require more volume at the beginning of the run to account for added evaporation. This volume should be determined empirically. A PCR plate is the most practical and common plate type at deck location 4 because of the low volumes of solution typically used at that location. The minimum volume required in the wells at the end of the incubation time for other plate types must be determined empirically.

Example using Red PCR Plate Insert plus PRC plate. To conduct a 30-minute reaction at a resin bed temperature of approximately 37 C with a 6 L total aspiration volume, the temperature should be set to 45 C and 12 L of enzyme solution should be added per well before the run is started. 12 L is required because 6 L will be aspirated, 3 L are expected to evaporate, and 3 L are required at the end of the run to ensure heat conductance occurs during the entire run. Temperature C: Default: 25 Practical: 2060 Range: 4110

Protocol step Guidelines and notes

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Duration This setting specifies the total length of time to aspirate the Reagent through the cartridges, including the initial and secondary aspiration steps. Note: This setting governs only the aspiration of Reagent through the cartridges. It does not include the time required to ramp up to temperature or the time required for the Reaction Chase step. Note: The duration of the reaction is related to the concentration of the reactant. In general, as the concentration of the reactant (for example, enzyme) decreases, the duration increases, as does the amount of reaction solution lost during the reaction due to evaporation, and the volume of excess reaction solution required. See the notes in the preceding Temperature step for more details. Time (minutes): Default: 30 Practical: 560 Range: 0180

Initial Draw Specifies the initial draw volume, which is aspirated at 10 L/min. Any additional volume (reaction volume minus the initial draw volume) is aspirated at a flow rate appropriate to satisfy the Duration setting. Volume (L): Volume for 5 L cartridge:

Default: 4 Practical: 39 Range: 0250

Volume for 25 L cartridge: Default: 20 Practical: 1545 Range: 0250

Note: Setting the volume to zero skips all Reaction tasks except syringe washing.

Protocol step Guidelines and notes

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Reaction Chase This step washes the soluble reaction products out of the resin bed and into the syringes using the Chase Buffer. The Reaction Chase occurs immediately after the aspiration of Reagent, combining the flow-through of the initial and secondary aspiration and the Chase Buffer within the syringes. Volume (L). Elution of products off the AssayMAP cartridges is typically 23 column volumes when the affinity between the eluate and resin has been completely disrupted. Therefore, 1015 L (5 L cartridge) or 5075 L (25 L cartridge) should be a sufficient chase volume as long as there is no significant affinity between the soluble reaction product and the resin bed. The default volume is set to 3 column volumes to be conservative, but it is likely that you can decrease this volume. Volume for 5 L cartridge:

Default: 15 Practical range: 1530 Range: 0250

Volume for 25 L cartridge: Default: 75 Practical range: 50100 Range: 0250

Note: Setting the volume to zero skips all Reaction tasks except syringe washing. Flow Rate: Default: 5 Practical: 520 Range: 0.1500

Combine with Eluate

This setting specifies where the combination of the Reaction initial and secondary aspiration and the Chase Buffer is collected: If selected, collects the Reaction initial and secondary aspiration and chase in the

Eluate Collection plate. This is a good choice if the downstream analysis can easily separate the reactant, soluble, and eluted reaction products, allowing all the reaction products to be analyzed in a single run.

If not selected, collects the Reaction initial and secondary aspiration and chase in the Flow Through Collection plate. This is a good choice if the goal is to analyze the soluble and immobilized reaction products separately because Data on only one of these components is required, and there is not a good or

rapid way of chromatographically separating the reactant, buffer, soluble, and immobilized reaction products in a single analytical run.

One wants to remove the buffer, reactant, and soluble reaction product from the immobilized reaction product, and elute this immobilized reaction product in a mass-spec-compatible solution.

The setting is not selected by default.

Protocol step Guidelines and notes

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Cup Wash 1 This step removes the residual liquid that may remain above the resin bed after the Reaction step. The Cup Wash 1 step aspirates Cartridge Wash Buffer 1 into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 1

This step uses Cartridge Wash Buffer 1 to wash non-specifically bound molecules from the resin bed. In preparation for Internal Cartridge Wash 1, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Cartridge Wash Buffer 1 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 1 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate. A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

This step is typically used for optimization or troubleshooting of a protocol to ensure that the wash solution is not eluting the immobilized substrate off the cartridge. If this step is selected, the flow-through from Internal Cartridge Wash 1 step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash 1 is dispensed into the wash station. This step is not selected by default.

Cup Wash 2 This step removes the residual buffer that may remain above the resin bed after the Internal Cartridge Wash 1 step. This step aspirates Cartridge Wash Buffer 2 and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the previous cartridge wash is aspirated from the cartridge cups. Any cartridges that stuck to the probes during the cup wash are parked at the seating station, and then the liquid in the syringes is dispensed into the wash station. The syringes are washed at the wash station. This step is not selected by default. Volume (L). A volume less than the default may be insufficient for cup washing, while a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash 2

This step uses Cartridge Wash Buffer 2 to wash non-specifically bound molecules and Cartridge Wash Buffer 1 from the resin bed. In preparation for Internal Cartridge Wash 2, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid that is in the cups is removed by a 60 L aspiration, the aspirated solution is discarded at the wash station, 10 L of Cartridge Wash Buffer 2 is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer 2 into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges at the specified flow rate into the Flow Through Collection plate or wash station. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer from the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. This step is not selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) has little benefit but will also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50150 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate. A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of deionized water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

This step is typically only used for optimization or troubleshooting of a protocol to ensure that the wash solution is not eluting the immobilized substrate off the cartridge. If this step is selected, the liquid eluted during Internal Cartridge Wash 2 is dispensed directly into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through is dispensed directly into the wash station. This step is not selected by default.

Stringent Syringe Wash

This step cleans the syringes with the Elution Buffer prior to elution. The Stringent Syringe Wash step aspirates the Elution Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the wash station. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

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Elute This step uses Elution Buffer to elute immobilized reaction products from the cartridges. In preparation for elution, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station, 10 L of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the eluate is mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 L, the samples will not be mixed. You can also select the Eluate Discard and Existing Collection Volume substeps, which are described in the following rows of this table. This step is selected by default. Volume (L). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges, or 5075 L for 25 L cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Note: The Eluate Collection plate must be able to accommodate the total volume, which is determined by summing the net elution volume (Elute volume - Eluate Discard volume), the Reaction and Reaction chase volumes if Combine With Eluate Volume is selected, and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Volume for 5 L cartridges:

Default: 25 Practical: 1030 Range: 0250

Volume for 25 L cartridges: Default: 125 Practical: 50150 Range: 0250

Note: Setting the volume to zero skips all Elute tasks except syringe washing.

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Flow rate. A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 215 Range: 0.1500 Wash cycle. The number of syringe washes to perform at the wash station after an Elute step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Eluate Discard This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. The Eluate Discard volume is dispensed into the wash station. The remaining Elution Buffer is dispensed through cartridges at the Elute flow rate into the Eluate Collection plate.

Example: If the Elute and Eluate Discard steps are selected with the following settings, Elute volume = 15 L (5 L cartridges) or 50 L (25 L cartridges) Eluate Discard volume = 2 L (5 L cartridges) or 10 L (25 L cartridges) the first 2 L (5 L cartridges) or 10 L (25 L cartridges) eluate from the cartridges will be discarded to the wash station, and the remaining 13 L (5 L cartridges) or 40 L (25 L cartridges) eluate will be collected in the Eluate Collection plate.

Select the Eluate Discard step in a situation where minimizing the volume of eluate is critical. For AssayMAP cartridges, the initial elution volume (~2 L for 5 L cartridges and ~10 L for 25 L cartridges) contains small or no measurable amounts of analyte. This step is not selected by default. Volume (L). The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. Default: 0 Practical:

5 L cartridges: 02 25 L cartridges: 010

Range: 0250

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Existing Collection Volume

This step enables you to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. The Existing Collection Volume, the net volume from the Elute step (Elute volume - Eluate Discard volume), and the soluble reaction products, if Combine With Eluate is selected, feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head into and out of the wells during elution and eluate mixing in a volume-dependent manner. For the maximum practical working volumes of labware for eluate collection, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (L): Default: 0 Practical: 0250 Range: 01000 Note: Total elution collection well volumes >500 L may require additional off-deck mixing to reach homogeneity.

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the wash station. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 L of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the On-Cartridge Reaction protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Start protocol Sets Peltier Thermal Station to set point temperature (C).

2 Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes the specified number of cycles.

Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then performs an external probe wash.

3 Aspirates 10 L of Equilibration Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Equilibration Buffer into the cartridge cups and exercises the cartridges off task.

3 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

1 Dispenses the Equilibration Buffer through the cartridges to equilibrate. Washes the cartridge exteriors.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

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Reaction 1 Waits for Peltier Thermal Station to reach 2 C of the Reaction Temperature setting.

2 Mounts the cartridges on the head.

4 Aspirates Initial Draw volume at 10 L/min.

4 Aspirates the remaining volume (Reaction Volume setting in the form minus Initial Draw volume) for the specified Duration (minutes) at the set Temperature (C).

1 Washes the cartridge exteriors at the wash station.

3 Aspirates the specified volume of Chase Buffer at the specified Flow Rate (L/min).

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

7 Dispenses the flow-through into the Flow Through Collection plate. Mixes the flow-through.

1 Washes the syringes.

Cup Wash 1 5 Aspirates cartridge Cartridge Wash Buffer 1 into the syringes.

2 Washes the cartridge cups and exercises the cartridges off task.

1 Dispenses the buffer into the wash station between the chimneys.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Internal Cartridge Wash 1

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then performs an external probe wash.

5 Aspirates 10 L of Cartridge Wash Buffer 1 for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

5 Aspirates Cartridge Wash Buffer 1 into the syringes for the Internal Cartridge Wash 1 step.

2 Mounts the cartridges on the head.

1 Dispenses Cartridge Wash Buffer 1 through the cartridges at the Internal Cartridge Wash 1 flow rate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Stringent Syringe Wash

8 Aspirates the Syringe Wash Buffer (Elution Buffer).

1 Dispenses the buffer at the wash station.

1 Washes the syringes.

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Elute 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys, and then performs an external probe wash.

8 Aspirates 10 L of Elution Buffer for the cartridge air-gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Elution Buffer.

2 Mounts the cartridges.

9 Elutes the samples into the Eluate Collection plate.

1 Washes the cartridge exteriors.

2 Parks the cartridges in the seating station.

9 Mixes eluates.

1 Washes the syringes.

Final Syringe Wash 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

1 Dispenses into the wash station between the chimneys.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Reference library

1 Han, J., Van Den Heuvel, Z., & Murphy, S. A streamlined drug-to-antibody ratio determination workflow for intact and deglycosylated antibody-drug conjugates, Agilent Technologies, Inc, September 2019, 5991-9010EN.

2 Wu, S., Shen, M., Murphy, S., & Van Den Heuvel, Z. An Integrated Workflow for Intact and Subunits of Monoclonal Antibody Accurate Mass Measurements. Agilent Technologies, Inc, March 2018, 5991-8445EN.

See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Bravo Platform.

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This chapter contains the following topics: App description on page 356 Before you start on page 356 Preparing the solutions on page 361 Preparing the samples on page 364 Running the protocol on page 368 Assay development guidelines and protocol notes on page 376 Reference library on page 392 To view the quick start guide for this application, see Quick start guides on page 1. Note: This section presents instructions for using the Peptide Cleanup v4.0 application. If you are using the Aspiration Mode version, see Peptide Cleanup: Aspiration Mode v3.0 User Guide on page 393.

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App description

Peptide Cleanup v4.0. This application enables automated cleanup of from 1 to 96 peptide samples in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Peptide Cleanup protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. You might need the following when preparing the samples and reagents:

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate vacuum concentrator for concentration and drying of samples Microplate sealer, such as the PlateLoc Sealer or equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Peptide Cleanup on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges: Use only primed and equilibrated cartridges.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Reversed-Phase (C18) cartridge rack 5190-6532 G5496-60017

AssayMAP Reversed-Phase (RP-S) cartridge rack G5496-60033 G5496-60023

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of C18 and RP-S 5- or 25-L cartridges are required. For details, see Preparing the samples on page 364.

G5496-60009

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IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

Starter kits

The following table lists the two starter kits that are available. Each starter kit contains both cartridges and labware.

The following table lists labware that are included in the Peptide Cleanup Starter Kit. Note: The labware included in the starter kits are for both the Peptide Cleanup and In- Solution Digestion applications. For the Peptide Cleanup labware requirements, see Labware on page 360. For the In-Solution Digestion: Multi-Plate labware requirements, see In-Solution Digestion: Multi-Plate v2.0 User Guide on page 235.

Starter Kit Part number

AssayMAP Digestion and RP-S Cleanup Starter Kit Contains 96 Reversed-Phase (RP-S) cartridges and Labware for In-Solution Digestion: Multi-Plate and Peptide Cleanup

G5496-60034

AssayMAP Digestion and C18 Cleanup Starter Kit Contains 96 Reversed-Phase (C18) cartridges and Labware for In-Solution Digestion: Multi-Plate and Peptide Cleanup

G5496-60013

Labware Part number

1.2 mL Deep-Well PolyPro Clear Plates (qty 2) ABgene AB-1127

96-Well U-Bottom PolyPro White Plates (qty 11) Greiner 650207

250-L Pipette Tips (qty 1) Agilent 19477-02

12-Column Low-Profile Reservoirs (qty 4) Agilent 201280-100

96-Well Round-Bottom, Clear Plates (qty 2) Greiner 650201

96-Well PCR Plates (qty 3) Eppendorf 30129300

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Labware

Labware requirements vary depending on experimental design. The following table provides a complete list labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware Manufacturer part number* Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3, 58

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 3, 58

96 Agilent, 2mL Square Deep Well labware Agilent 204353-100 3, 7

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 38

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7, 9**

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 4, 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 5, 6, 8

*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner PCR plate is not compatible with the 25 L cartridges at deck locations 7 and 9.

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Reagents

The volume, type, and concentration of reagents required for peptide cleanup vary depending on sample characteristics and the desired analytical result. Consult published literature for reagent recommendations for sample and surface chemistry combinations. See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Peptide Cleanup application. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 361.

Preparing the solutions

The following solutions are required for the Peptide Cleanup protocol: Priming & Syringe Wash Buffer Equilibration & Cartridge Wash Buffer Elution Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Peptide Cleanup

The Reagent Volume Calculator is a Microsoft Excel file that contains the following: Calculator worksheet. You enter the number of columns to process, whether to

perform the Collect Flow Through option, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

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Reagent Recipe worksheet. You enter the concentrations of each component in your reagent, and the worksheet calculates the recipe volumes required.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figures show the worksheets of the Reagent Volume Calculator.

Figure Peptide Cleanup Reagent Volume Calculator worksheet

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Figure Peptide Cleanup Reagent Recipe worksheet

Preparing the buffers

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the AssayMAP Peptide Cleanup chemistry.

Table Reagent preparation

Reagent (deck location) Composition and comments

Priming & Syringe Wash Buffer (deck location 5)

Typically 50% or greater organic solution (must be >25% organic), acidic, and identical in composition to the Elution Buffer to simplify the solution preparation, for example, 70% ACN: 29.9% H2O : 0.1% TFA

The high-percentage organic composition is essential for reversed-phase cartridges as they ship dry and must be wetted with a high percentage organic solution or they will have very little binding capacity. This solution, the other solutions used in this application, and the sample are typically acidic. Other more mass-spec-friendly acids can substitute for TFA.

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Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and cartridge positions on the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 364.

To dispense the solutions into the labware: 1 Optional. Label each piece of labware so that you can easily identify them. 2 Add the specified volume of the Priming Buffer & Syringe Wash Buffer into the

labware to be placed at deck location 5. 3 Add the specified volume of Elution Buffer into the labware to be placed at deck

location 6. 4 Add the specified volume of Equilibration & Cartridge Wash Buffer into the labware

to be placed at deck location 8. 5 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Peptide Cleanup protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must:

Elution Buffer (deck location 6)

High percentage organic and acidic solution, for example, 70% ACN: 29.9% H2O : 0.1% TFA

Equilibration & Cartridge Wash Buffer (deck location 8)

Very low or no organic and acidic solution. Typically, similar in the percentage organic and acidity to the sample, for example, 99.9% H2O : 0.1% TFA

Reagent (deck location) Composition and comments

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Remove macromolecular particulates before the samples are loaded onto AssayMAP cartridges.

Adjust the buffer composition to the optimal binding conditions (for example, low pH and low organic).

Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the buffer composition

One of the most important considerations for highly efficient and unbiased binding of peptides to the reversed-phase resin is the pH of the sample, which should be acidic for standard, low-pH peptide cleanup with C18 or RP-S cartridges or basic with RP-S cartridges. Peptides are amphoteric molecules with a diverse range of physiochemical properties. For some classes of peptides, cleanup under high-pH conditions may help to promote retention (e.g., highly basic peptides), or preserve acid-labile modifications (e.g., histidine phosphorylation). You can acidify samples for low-pH peptide cleanup before loading onto AssayMAP C18 and RP-S cartridges generally by adding TFA, formic acid, or acetic acid. Sample pH for high-pH peptide cleanup is commonly adjusted using aqueous solutions of ammonium formate or ammonium acetate titrated to a pH > 10 using ammonium hydroxide. You can use the Reagent Transfer utility to perform these pH adjustments. For instructions, see Reagent Transfer v3.0 User Guide on page 525. Samples containing organic solvents or some types of detergents should be avoided as they might bias the peptides that bind the column. For example, loading samples in a buffer containing greater than 5% acetonitrile will inhibit binding of hydrophilic peptides. Silica-based C18 cartridges are at risk with a sample pH higher than 8. If you have concerns about a specific buffer component, you should examine scientific literature for the known effects of this type of molecule on reversed-phase resins. You can use the Reagent Transfer utility to modify the composition of your samples with a dilution or pH adjustment, especially after using the In-Solution Digestion workflow.

Determining the volume of sample to load

The AssayMAP Peptide Cleanup protocol permits loading up to 1000 L of sample onto AssayMAP cartridges. For sample volumes > 250 L, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 L) of the Bravo 96AM Head.

What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity:

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Quantitative binding capacity. The maximum mass of peptide that can bind to the cartridge in a single pass, where less than 10% of the load appears in the flow- through. For a single species of peptide, the quantitative binding capacity is relatively straightforward. The quantitative binding capacity for a mixture of peptides is more complex due to the differences in relative hydrophobicity of the peptides, which results in competitive binding in situations where the ratio of binding sites to mass loaded is low. Examples of how sample mass loaded and the relative hydrophobicity of peptides can affect recovery in a complex peptide mixture, see Agilent app note 5991- 2957EN in the Reference library on page 392. To avoid or minimize sample bias during peptide cleanup, it is critical to load sample masses that are less than the mass at which hydrophillic peptides are lost.

Total binding capacity. The maximum mass of peptide that can bind to the cartridge. This can only be achieved by loading significantly more peptide than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity and will result in the loss of hydrophillic peptides.

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for the 5 and 25 L RPS and C18 cartridges.

What is the concentration of the target in the sample? If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows.

The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the Bravo Platform applies differential pressure to seat cartridges based upon the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use the AssayMAP Resin- Free cartridges (Agilent part number G5496-60009) to fill the empty column and row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash step.

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Figure Example of sample microplate and reservoir layout: A) Multiple of 8 samples, B) Not a multiple of 8

See Labware on page 360 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well or column does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

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Running the protocol

The Peptide Cleanup protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the peptides from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the cartridge seating station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 361. Prepare the samples. See Preparing the samples on page 364.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See System Startup/Shutdown v3.0 User Guide.

Transfer the cartridges to the 96AM Cartridge & Tip Seating Station. See Cartridge Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent Technologies does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 358.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Peptide Cleanup application.

To set up the protocol: 1 Open the App Library. 2 Locate Peptide Cleanup, and then click App.

The Peptide Cleanup application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 374.

To modify the method, proceed to step 6. VWorks Plus. Only VWorks administrators or technicians may modify and save methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

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9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 376.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 5) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

Equilibrate Aspirates Equilibration Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates samples (deck location 4) into the syringes, and then dispenses them through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 7).

5 L: 100 5 3

25 L: 100 5 3

Range: 01000 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the sample flow-through in the Organic Waste plate (deck location 3).

Cup Wash Rinses the cartridge cups with Cartridge Wash Buffer (deck location 8), and then discards the liquid into the Organic Waste plate (deck location 3).

5 L: 25 3

25 L: 25 3

Range: 0100 010

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Internal Cartridge Wash

Aspirates Cartridge Wash Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 7).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the Internal Cartridge Wash flow-through in the Organic Waste plate (deck location 3).

Stringent Syringe Wash

Aspirates Syringe Wash Buffer (deck location 5) into the syringes, and then dispenses it into the Organic Waste plate (deck location 3).

5 L: 50 2

25 L: 50 2

Range: 0250 010

Elute Aspirates Elution Buffer (deck location 6) into the syringes, and then dispenses it through the cartridges into the Eluate Collection plate (deck location 9).

5 L: 25 5 1

25 L: 125 5 1

Range: 0250 0.1500 010

Eluate Discard

If selected, a specified initial volume of the eluate is discarded into the Organic Waste plate (deck location 3), or collected in the Flow Through Collection plate (deck location 7).

5 L: 0

25 L: 0

Range: 0250

Add to Flow Through

If selected, collects the Eluate Discard in the Flow Through Collection plate (deck location 7). If not selected, discards the Eluate Discard into the Organic Waste plate (deck location 3).

Existing Collection Volume

Specifies the volume of liquid present in the Eluate Collection plate (deck location 9) at the beginning of the run.

5 L: 0

25 L: 0

Range: 0300

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

**For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 377. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 388.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error messages if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner PCR plates are not compatible with the 25 L cartridges at deck locations 7 and 9.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

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2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

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Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Peptide Cleanup protocol to

your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

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Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply too

much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

Initial Syringe Wash

This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

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Prime This step removes entrained air from the packed resin bed and properly wets the surface of the resin. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) used with the Peptide Cleanup application require a Priming Buffer containing at least 25% organic solvent. As illustrated in Agilent app note 5991-6478EN (Reference library on page 392), reversed- phase cartridges (C18, RPS, and RPW) that are primed with solutions containing a low percentage of organic solvent have greatly reduced binding capacity. Agilent recommends using a priming solution that contains at least 50% of an organic solvent such as acetonitrile. This step is selected by default. Volume (L). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridges:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow Rate (L/min): A flow rate lower than the default value diminishes the ability to effectively remove entrained air from the cartridge. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5500 Wash Cycles: The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the Organic Waste plate The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) typically used with the Peptide Cleanup application require an equilibration solution that has a very low concentration of or no organic solvent for effective binding during the sample loading step. This step is selected by default. Volume (L). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow Rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min using the default volume may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash Cycles: The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes, and then performs an external syringe wash at the wash station to remove any sample remaining on the outside of the probes before mounting the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. The protocol accommodates sample volumes up to 1000 L to be dispensed through AssayMAP peptide cleanup cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L. Each syringe has a maximum capacity of 250 L. When sample volumes are greater than 250 L, the protocol will iteratively load samples onto cartridges. To determine the number and the volume of iterative loads, the protocol uses the following formulas: # of times to load = Total sample volume/250,

and the result is rounded up to nearest integer Volume of each load = Sample volume/# of times to load For example, if the total sample volume is 900 L, then:

# times to load = 900/250 = 3.6, which is rounded up to 4 Volume of each load = 900/4 = 225

If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

IMPORTANT Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For more information, see Transferring the samples to the microplate on page 367.

To determine the volume of sample to load, see Determining the volume of sample to load on page 365. This step is selected by default.

Protocol step Guidelines and notes

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Volume (L). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. Large sample volumes (> 250 L) may require slightly more excess sample due to evaporation. Default: 100 Practical: 101000 Range: 01000 Note: The lower the sample volume the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed in the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed in the Organic Waste plate. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.

Protocol step Guidelines and notes

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Cup Wash This step removes the residual sample liquid that may remain above the resin bed after the Load Samples step. The Cup Wash step aspirates Cartridge Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Organic Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (L): Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash Cycles: Each wash cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Internal Cartridge Wash

This step uses Cartridge Wash Buffer to wash non-specifically bound molecules from the resin bed. In preparation for the Internal Cartridge Wash, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Cartridge Wash Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates the Cartridge Wash Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station If the Load Samples step is selected, the first 5 L (5 L cartridges) or 25 L (25 L cartridges) of Cartridge Wash Buffer is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash volume minus the chase volume is dispensed at the Internal Cartridge Wash flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow Rate (L/min): A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

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Collect Flow Through

If this step is selected, the flow-through from the Internal Cartridge Wash step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from the Internal Cartridge Wash step is dispensed into the Organic Waste plate. This step is not selected by default.

Stringent Syringe Wash

This step cleans the syringes with the Syringe Wash Buffer prior to elution. The Stringent Syringe Wash step aspirates the Syringe Wash Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the Organic Waste plate. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash Cycles. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

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Elute This step uses Elution Buffer to elute bound peptides from the cartridges. In preparation for elution, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the samples are mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 L, the samples will not be mixed. You can also select the Eluate Discard and Add to Flow Through substeps, which are described in the following rows of this table. This step is selected by default. Volume (L): The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges, or 5075 L for 25 L cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Note: The Eluate Collection plate must be able to accommodate the total volume, which is determined by summing the net elution volume (Elute volume - Eluate Discard volume) and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Volume for 5 L cartridges:

Default: 25 Practical: 1030 Range: 0250

Volume for 25 L cartridges: Default: 125 Practical: 50150 Range: 0250

Note: Setting the volume to zero skips all Elute tasks except syringe washing.

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Elute (continued)

Flow Rate (L/min): A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/min for a given volume of elution buffer (that is, more buffer may be required to get the same elution yield at high flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500 Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Eluate Discard This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. If the Eluate Discard step is selected, the specified volume is dispensed into the Organic Waste plate or Flow Through Collection plate (if the Add to Flow Through step is selected). The remaining Elution Buffer is dispensed through cartridges at the Elute flow rate into the Eluate Collection plate.

Example: If the Elute, Eluate Discard, and Add to Flow Through steps are all selected with the following settings: Elute volume = 15 L (5 L cartridges) or 40 L (25 L cartridges) Eluate Discard volume = 2 L (5 L cartridges) or 10 L (25 L cartridges) the first 2 L (5 L cartridges) or 10 L (25 L cartridges) eluate from the cartridges will be discarded into the Flow Through Collection plate, and the remaining 13 L (5 L cartridges) or 30 L (25 L cartridges) eluate will be collected in the Eluate Collection plate.

Select the Eluate Discard step in situations where minimizing the volume of eluate is important. For AssayMAP cartridges, the initial elution volume (~2 L for 5 L cartridges or ~10 L for the 25 L cartridges) contains small or no measurable amounts of analyte. This step is not selected by default. Volume (L). The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. Default: 0 Practical:

5 L cartridges: 02 25 L cartridges: 010

Range: 0250

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Add to Flow Through

If selected, this step dispenses the Eluate Discard volume into the Flow Through Collection plate. If the Add to Flow Through step is not selected, the Eluate Discard is dispensed into the Organic Waste plate. This step is not selected by default. Note: The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected.

Existing Collection Volume

This step enables you to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. The Existing Collection Volume and the net volume from the Elute step (Elute volume - Eluate Discard volume) feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head into and out of the wells during elution and eluate mixing in a volume-dependent manner. For the maximum practical working volumes of the labware for the Eluate Collection plate, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, for adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (L): Default: 0 Practical: 0250 Range: 0300

Final Syringe Wash

This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 L of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L of DI water into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. Note: In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Peptide Cleanup protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Starting protocol 2 Parks all cartridges that might have been loaded on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes.

Prime 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

5 Aspirates 10 L of Priming Buffer for the cartridge air-gap prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

5 Aspirates the Priming Buffer.

2 Mounts the cartridges onto the head.

3 Dispenses the Priming Buffer through the cartridges and into Organic Waste plate to prime the cartridges.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

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Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

8 Aspirates 10 L of Equilibration Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Equilibration Buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Equilibration Buffer through the cartridges to equilibrate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Load Samples 4 Aspirates the samples into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

7 Dispenses the samples through the cartridges and into the Flow Through Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 8 Aspirates the Cartridge Wash Buffer.

2 Washes the cartridge cups and exercises the cartridges off task.

3 Dispenses the Cartridge Wash Buffer into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Internal Cartridge Wash

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

8 Aspirates 10 L of Cartridge Wash Buffer for the cartridge air- gap prevention step.

2 Dispenses the remaining 10 L of buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Cartridge Wash Buffer into the syringes for sample chase and the Internal Cartridge Wash steps.

2 Mounts the cartridges on the head.

3 Dispenses 5 L (5 L cartridges) or 25 L (25 L cartridges) Cartridge Wash Buffer through the cartridges at the Load Samples flow rate for the sample chase step.

3 Dispenses the remaining Cartridge Wash buffer through the cartridges at the Internal Cartridge Wash flow rate and into the Organic Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Stringent Syringe Wash

5 Aspirates the Syringe Wash Buffer.

3 Dispenses the Syringe Wash Buffer into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Elute 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

6 Aspirates 10 L of Elution Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Elution Buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates the Elution Buffer.

2 Mounts the cartridges on the head.

9 Elutes the samples into the Eluate Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

9 Mixes the eluates.

1 Washes the syringes.

Final Syringe Wash 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Reference library

1 Russel, J., Van Den Heuvel, Z., Bovee, M. & Murphy, S., Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-2957EN, 2016

2 Bovee, M., Krahenbuhl, A. & Murphy, S., Rapid Antibody Digestion Enabled by Automated Reversed-Phase Desalting on the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-6478EN, 2016

See the Agilent AssayMAP Bravo Citation Index for published papers that used the AssayMAP Bravo Platform.

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This chapter contains the following topics: App description on page 394 Before you start on page 394 Preparing the solutions on page 399 Preparing the samples on page 402 Running the protocol on page 405 Assay development guidelines and protocol notes on page 413 Reference library on page 423 Note: This section presents instructions for using the Peptide Cleanup: Aspiration Mode application. If you are using the Dispense Mode version, see Peptide Cleanup v4.0 User Guide on page 355.

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App description

Peptide Cleanup: Aspiration Mode v3.0. This application enables automated cleanup of from 1 to 96 peptide samples in a single run. This application aspirates the sample and wash solutions up through the cartridge resin bed rather than dispensing them through the resin bed, which is how the standard Peptide Cleanup application functions. For most customers, Agilent recommends using the standard Peptide Cleanup application instead of the Peptide Cleanup: Aspiration Mode application. Although the two applications yield similar results, the standard Peptide Cleanup application is less sensitive to clogging and, therefore, more robust. However, some customers find the Peptide Cleanup: Aspiration Mode application provides slightly better purification.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Peptide Cleanup: Aspiration Mode protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Note: The 25-L have not yet been optimized on the Peptide Cleanup: Aspiration Mode application. If you are interested in trying them on this application, contact Agilent Customer Service for advice.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Optional equipment. You might need the following when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate vacuum concentrator for concentration and drying of samples Microplate sealer, such as the PlateLoc Sealer or equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Peptide Cleanup on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges. Note: This application has not yet been optimized for the 25 L cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges:

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Part number

AssayMAP Reversed-Phase (C18) cartridge rack 5190-6532

AssayMAP Reversed-Phase (RP-S) cartridge rack G5496-60033

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or to fill remaining positions in columns that are partially occupied by the C18 and RP-S cartridges. For details, see Preparing the samples on page 402.

G5496-60009

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Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

Starter kits

The following table lists the two starter kits that are available. Each starter kit contains both cartridges and labware.

The following table lists labware that are included in the Peptide Cleanup Starter Kit. Note: The labware included in the starter kits are for both the Peptide Cleanup: Aspiration Mode and the In-Solution Digestion applications. For the Peptide Cleanup: Aspiration Mode labware requirements, see Labware on page 398. For the In-Solution Digestion: Multi-Plate labware requirements, see In-Solution Digestion: Multi-Plate v2.0 User Guide on page 235.

Starter Kit Part number

AssayMAP Digestion and RP-S Cleanup Starter Kit Contains 96 Reversed-Phase (RP-S) cartridges and Labware for In-Solution Digestion: Multi-Plate and Peptide Cleanup.

G5496-60034

AssayMAP Digestion and C18 Cleanup Starter Kit Contains 96 Reversed-Phase (C18) cartridges and Labware for In-Solution Digestion: Multi-Plate and Peptide Cleanup.

G5496-60013

Labware Part number

1.2 mL Deep-Well PolyPro Clear Plates (qty 2) ABgene AB-1127

96-Well U-Bottom PolyPro White Plates (qty 11) Greiner 650207

250-L Pipette Tips (qty 1) Agilent 19477-02

12-Column Low-Profile Reservoirs (qty 4) Agilent 201280-100

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Labware

Labware requirements vary depending on experimental design. The following table provides a complete list labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

96-Well Round-Bottom, Clear Plates (qty 2) Greiner 650201

96-Well PCR Plates (qty 3) Eppendorf 30129300

Labware Part number

Labware Manufacturer part number* Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3, 58

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 58

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 38

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7, 9

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 4, 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 58

*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Reagents

The volume, type, and concentration of reagents required for peptide cleanup vary depending on sample characteristics and the desired analytical result. Consult published literature for reagent recommendations for sample and surface chemistry combinations. See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Peptide Cleanup application. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 399.

Preparing the solutions

The following solutions are required for the Peptide Cleanup: Aspiration Mode protocol: Priming & Syringe Wash Buffer Equilibration & Cartridge Wash Buffer Elution Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Peptide Cleanup: Aspiration Mode

The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, whether to perform the Collect Flow Through options, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

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To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click Restore Defaults. 5 Modify the values in the green boxes as required to match your specific method. As

you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figure shows the worksheet of the Reagent Volume Calculator.

Figure Reagent Volume Calculator for Peptide Cleanup: Aspiration Mode

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Preparing the buffers

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the AssayMAP Peptide Cleanup: Aspiration Mode chemistry.

Table Reagent preparation

Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and cartridge positions on the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 402.

To dispense the solutions into the labware: 1 Optional. Label each piece of labware so that you can easily identify them. 2 Add the specified volume of the Priming Buffer & Syringe Wash Buffer into the

labware to be placed at deck location 5.

Reagent (deck location) Composition and comments

Priming & Syringe Wash Buffer (deck location 5)

Typically 50% or greater organic solution (must be >25% organic), acidic, and identical in composition to the Elution Buffer to simplify the solution preparation, for example, 70% ACN: 29.9% H2O : 0.1% TFA

The high-percentage organic composition is essential for reversed-phase cartridges as they ship dry and must be wetted with a high percentage organic solution or they will have very little binding capacity. This solution, the other solutions used in this application, and the sample are typically acidic. Other more mass-spec-friendly acids can substitute for TFA.

Elution Buffer (deck location 6)

High percentage organic and acidic solution, for example, 70% ACN: 29.9% H2O : 0.1% TFA

Equilibration & Cartridge Wash Buffer (deck location 8)

Very low or no organic and acidic solution. Typically, similar in the percentage organic and acidity to the sample, for example, 99.9% H2O : 0.1% TFA

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3 Add the specified volume of Elution Buffer into the labware to be placed at deck location 6.

4 Add the specified volume of Equilibration & Cartridge Wash Buffer into the labware to be placed at deck location 8.

5 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Peptide Cleanup: Aspiration Mode protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to the optimal binding conditions (for example, low

pH and low organic). Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the buffer composition

One of the most important considerations for highly efficient and unbiased binding of peptides to the reversed-phase resin is the pH of the sample, which should be acidic for standard, low-pH peptide cleanup with C18 or RP-S cartridges or basic with RP-S cartridges. Peptides are amphoteric molecules with a diverse range of physiochemical properties. For some classes of peptides, cleanup under high-pH conditions may help to promote retention (e.g., highly basic peptides), or preserve acid-labile modifications (e.g., histidine phosphorylation). You can acidify samples for low-pH peptide cleanup before loading onto AssayMAP C18 and RP-S cartridges generally by adding TFA, formic acid, or acetic acid. Sample pH for high-pH peptide cleanup is commonly adjusted using

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aqueous solutions of ammonium formate or ammonium acetate titrated to a pH > 10 using ammonium hydroxide. You can use the Reagent Transfer utility to perform these pH adjustments. For instructions, see Reagent Transfer v3.0 User Guide on page 525. Samples containing organic solvents or some types of detergents should be avoided as they might bias the peptides that bind the column. For example, loading samples in a buffer containing greater than 5% acetonitrile will inhibit binding of hydrophilic peptides. Silica-based C18 cartridges are at risk with a sample pH higher than 8. If you have concerns about a specific buffer component, you should examine scientific literature for the known effects of this type of molecule on reversed-phase resins. You can use the Reagent Transfer utility to modify the composition of your samples with a dilution or pH adjustment, especially after using the In-Solution Digestion workflow.

Determining the volume of sample to load

The AssayMAP Peptide Cleanup protocol permits loading up to 1000 L of sample onto AssayMAP cartridges. For sample volumes > 250 L, the protocol will iteratively load samples onto cartridges to stay within the maximum syringe volume (250 L) of the Bravo 96AM Head.

What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity: Quantitative binding capacity. The maximum mass of peptide that can bind to the

cartridge in a single pass, where less than 10% of the load appears in the flow- through. For a single species of peptide, the quantitative binding capacity is relatively straightforward. The quantitative binding capacity for a mixture of peptides is more complex due to the differences in relative hydrophobicity of the peptides, which results in competitive binding in situations where the ratio of binding sites to mass loaded is low. Examples of how sample mass loaded and the relative hydrophobicity of peptides can affect recovery in a complex peptide mixture, see Agilent app note 5991- 2957EN in the Reference library on page 423. To avoid or minimize sample bias during peptide cleanup, it is critical to load sample masses that are less than the mass at which hydrophillic peptides are lost.

Total binding capacity. The maximum mass of peptide that can bind to the cartridge. This can only be achieved by loading significantly more peptide than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity and will result in the loss of hydrophillic peptides.

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for the RPS and C18 cartridges.

What is the concentration of the target in the sample? If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

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Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows.

The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the Bravo Platform applies differential pressure to seat cartridges based upon the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use the AssayMAP Resin- Free cartridges (Agilent part number G5496-60009) to fill the empty column and row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash step.

Figure Example of sample microplate and reservoir layout

See Labware on page 398 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

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An excess volume (overage) ensures that the sample does not fully deplete. Aspirating from depleted wells can cause air to enter the cartridges, thus compromising results. Modify this excess volume accordingly if evaporation of volatile solvents is a concern. See the Labware Reference Guide for labware-specific overage recommendations. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs. Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility to transfer the samples. For instructions, see

Reagent Transfer v3.0 User Guide on page 525. 2 If necessary, centrifuge the sample labware to remove bubbles.

Running the protocol

The Peptide Cleanup: Aspirate Mode protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the peptides from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the cartridge seating station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

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Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 399. Prepare the samples. See Preparing the samples on page 402. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs. Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide. Transfer the cartridges to the 96AM Cartridge & Tip Seating Station. See Cartridge

Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 396.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Peptide Cleanup: Application Mode application.

To set up the protocol: 1 Open the App Library. 2 Locate Peptide Cleanup: Aspiration Mode, and then click App.

The Peptide Cleanup: Aspiration Mode application opens.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 410. To modify the method, proceed to step 6. VWorks Plus. Only VWorks administrators or technicians may modify and save methods.

6 In the Application Settings area, specify the Number of Full Columns of Cartridges. Note: The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

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Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 413.

Table Application Settings overview

Steps* Description Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 5) into the syringes, and then dispenses it through the cartridges into the Organic Waste (deck location 3).

5 L: 100 300

Range: 100 300

Equilibrate Aspirates Equilibration Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the wash station (deck location 1).

5 L: 50 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates up to 245 L of samples (deck location 4) through the mounted cartridges into the syringes, performs an external cartridge tip wash at the wash station (deck location 1), and then aspirates a 5-L chase of Equilibration Buffer (deck location 8). The cartridges are removed (deck location 2) and then the flow- through is dispensed into Flow Through Collection (deck location 7). Samples >245 L are loaded in multiple steps.

5 L: 100 5 3

Range: 0245 0.1500 010

Cup Wash Rinses the cartridge cups with Cartridge Wash Buffer (deck location 8), and then discards the liquid into the wash station (deck location 1).

5 L: 25 3

Range: 0100 010

Internal Cartridge Wash

Aspirates Cartridge Wash Buffer (deck location 8) through the cartridges, removes the cartridges from the probes, dispenses the contents of the syringes into the wash station (deck location 1).

5 L: 50 10 3

Range: 0250 0.5500 010

Stringent Syringe Wash

Aspirates Syringe Wash Buffer (deck location 5) into the syringes, and then dispenses the buffer into Organic Waste (deck location 3).

5 L: 50 2

Range: 0250 010

Elute Aspirates Elution Buffer (deck location 6) into the syringes, and then dispenses the buffer through the cartridges into the Eluate Collection plate (deck location 9).

5 L: 25 5 1

Range: 0250 0.1500 010

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error messages if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

Re- Equilibrate

Aspirates Equilibration Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into waste at the wash station (deck location 1).

5 L: 50 10

Range: 0250 0.5500

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 413. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 421.

Steps* Description Volume (L)

Flow Rate (L/min)

Wash Cycles

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CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

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2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

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Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Peptide Cleanup: Aspiration

Mode protocol to your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply too much

force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

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Initial Syringe Wash

This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

Prime This step removes entrained air from within the cartridges and properly wets the surface of the resin. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) used with the Peptide Cleanup application require a Priming Buffer containing at least 25% organic solvent. As illustrated in Agilent app note 5991-6478EN (Reference library on page 423), reversed-phase cartridges (C18, RPS, and RPW) that are primed with solutions containing a low percentage of organic solvent have greatly reduced binding capacity. Agilent recommends using a priming solution that contains at least 50% of an organic solvent such as acetonitrile. This step is selected by default. Volume (L): Default: 100 Range: 100 Flow Rate (L/min): Default: 300 Range: 300

Protocol step Guidelines and notes

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) typically used with the Peptide Cleanup application require an equilibration solution that has a very low concentration of or no organic solvent for effective binding during the sample loading step. This step is selected by default. Volume (L). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow Rate (L/min). A flow rate slower than the default will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min using the default volume may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash Cycles: The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. This step mounts the cartridges on the syringes, aspirates the samples through the cartridges, and performs an external cartridge tip wash at the wash station to remove any sample on the outside of the cartridge tips. Then 5 L of Equilibration Buffer (sample chase) is aspirated through the cartridges at the Load Samples flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. The cartridges are parked at the seating station and the flow-through plus sample chase are collected in the Flow Through Collection plate. The protocol accommodates sample volumes up to 245 L. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L.

IMPORTANT Be sure to include the recommended labware-dependent volume overage to prevent air from entering the cartridge. For more information, see Transferring the samples to the microplate on page 404.

This step is selected by default. Volume (L). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. To determine the volume of sample to load, see Determining the volume of sample to load on page 403. Default: 100 Practical: 25245 Range: 0245 Note: The lower the sample volume the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. Default: 5 Practical: 215 Range: 0.1500 Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Protocol step Guidelines and notes

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Cup Wash This step removes the residual sample liquid that may remain above the resin bed after the Load Samples step. The Cup Wash step aspirates Cartridge Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station at an offset from the chimneys, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash Cycles: Each wash cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

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Internal Cartridge Wash

This step uses Cartridge Wash Buffer to wash non-specifically bound molecules from the resin bed. For the wash operation, this step mounts the cartridges, aspirates Cartridge Wash Buffer through the cartridges, washes the exterior of the cartridges, parks the cartridges, and then dispenses the contents of the syringes into the wash station at an offset from the chimneys. The syringes are then washed at the wash station. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow Rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Stringent Syringe Wash

This step cleans the syringes with the Syringe Wash Buffer prior to elution. The Stringent Syringe Wash step aspirates the Syringe Wash Buffer and then dispenses the buffer into the Organic Waste plate. The syringes are then washed at the wash station. This step is selected by default. Volume (L): Volumes higher than the default volume may improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash Cycles. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Elute This step uses Elution Buffer to elute bound peptides from the cartridges. The Elute step aspirates the Elution Buffer into the syringes, washes the probes at the wash station, and then mounts the cartridges and dispenses the buffer through the cartridges at the specified flow rate into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the samples are mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 L, the samples will not be mixed. This step is selected by default. Volume (L). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. The default volume is conservative and significantly higher than the minimum expected with a strong Elution Buffer. Default: 25 Practical: 1030 Range: 0250 Note: Setting the volume to zero skips all Elute tasks except syringe washing. Flow Rate (L/min). A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/min for a given volume of elution buffer (that is, more buffer may be required to get the same elution yield at high flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500 Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Re-Equilibrate This step uses Equilibration Buffer to return the cartridge to a low percentage organic solvent condition. The step aspirates the buffer into the syringes, mounts the cartridges, dispenses the buffer through the cartridges into the wash station, and then parks the cartridges. Volume (L). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Re-Equilibrate tasks except syringe washing. Flow Rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min using the default volume may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500

Final Syringe Wash

This step uses the wash station to flush potential contaminants from the syringes. During each Final Syringe Wash cycle, the head aspirates 250 L of DI water into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. Note: In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Peptide Cleanup: Aspiration Mode protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Start protocol 2 Parks all cartridges that might be on the head from a previously aborted protocol.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes.

Prime 5 Aspirates the Priming Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Priming Buffer through the cartridges and into the Organic Waste plate to prime the cartridges.

2 Parks the cartridges.

1 Washes the exterior of the syringe probes.

Equilibrate 8 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

1 Dispenses the Equilibration Buffer through the cartridges to equilibrate.

1 Washes the exterior of the cartridges.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Load Samples 2 Mounts the cartridges on the head.

4 Aspirates the samples through the cartridges into the syringes.

1 Washes the exterior of the cartridge tips.

8 Aspirates 5 L of Equilibration Buffer for the sample chase.

2 Parks the cartridges in the seating station.

7 Dispenses the flow-through plus sample chase into the Flow Through Collection plate.

1 Washes the syringes.

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Cup Wash 8 Aspirates the Cartridge Wash Buffer.

2 Washes the cartridge cups and exercises the cartridges off task.

1 Dispenses the Cartridge Wash Buffer into the wash station.

1 Washes the syringes.

Internal Cartridge Wash

2 Mounts the cartridges on the head.

8 Aspirates the Cartridge Wash Buffer through the cartridges into the syringes.

1 Washes the exterior of the cartridges.

2 Parks the cartridges.

1 Dispenses the Cartridge Wash Buffer into the wash station, and then washes the exterior of the syringe probes.

Stringent Syringe Wash

5 Aspirates the Syringe Wash Buffer.

3 Dispenses the syringe contents into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

Elute 6 Aspirates the Elution Buffer into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

9 Elutes the samples into the Eluate Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

9 Mixes the eluates.

1 Washes the syringes.

Re-Equilibrate 8 Aspirates Equilibration Buffer into the syringes.

2 Mounts the cartridges on the head.

1 Dispenses the Equilibration Buffer through the cartridges to equilibrate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

Final Syringe Wash 1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Reference library

1 Russel, J., Van Den Heuvel, Z., Bovee, M. & Murphy, S., Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-2957EN, 2016

2 Bovee, M., Krahenbuhl, A. & Murphy, S., Rapid Antibody Digestion Enabled by Automated Reversed-Phase Desalting on the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-6478EN, 2016

See the Agilent AssayMAP Bravo Citation Index for published papers that used the AssayMAP Bravo Platform.

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App description

Phosphopeptide Enrichment v3.0. This application enables automated phosphopeptide enrichment on from 1 to 96 samples in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Phosphopeptide Enrichment protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which is required for running the AssayMAP protocols.

Figure AssayMAP Bravo Platform components

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CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide. Optional equipment. You might need the following when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate vacuum concentrator for concentration and drying of samples Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

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For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Phosphopeptide Enrichment on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges: Use only primed and equilibrated cartridges.

IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

AssayMAP TiO2 cartridges are stable from approximately pH 1 to 14.

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Fe(III)-NTA cartridge rack G5496-60085

AssayMAP TiO2 cartridge rack G5496-60016

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or to fill remaining positions in columns that are partially occupied by Fe(III)-NTA or TiO2 cartridges, if necessary. For details, see Preparing the samples on page 434.

G5496-60009

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AssayMAP Fe(III)-NTA cartridges are stable from approximately pH 2 to 11. At levels approximately > pH 3.5, resin within the cartridges may turn from pale yellow to golden yellow to orange or brown due to the formation of iron(III) complexes (most commonly with hydroxide). Resin coloration and intensity are a function of both the pH and the chemical nature of the solutions passed through the cartridge. Bare NTA cartridges are stable from approximately pH 2 to 14.

AssayMAP Fe(III)-NTA cartridges that have been stripped (bare NTA cartridges) should be charged and used immediately.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware Manufacturer part number*

Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3, 58

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 3, 58

96 Agilent, 2mL Square Deep Well labware Agilent 204353-100 3, 7

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 38

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7, 9**

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 4, 7, 9**

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

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Reagents

The volume, type, and concentration of reagents and buffers required for phosphopeptide enrichment will vary depending on the specific AssayMAP phosphopeptide enrichment cartridge being used, the sample characteristics, and the desired analytical result. Consult Agilent app note 5991-6073EN (Reference library on page 462) for reagent recommendations for using the AssayMAP Fe(III)-NTA with the Phosphopeptide Enrichment application. The reference library also contains references to published papers for using both Fe(III)-NTA and TiO2 for phosphopeptide enrichment. See the Agilent AssayMAP Bravo Citation Index for published papers that use the Phosphopeptide Enrichment application on the AssayMAP Bravo Platform to purify phosphopeptides from a wide range of sample matrices. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 430.

Preparing the solutions

The following solutions are required for the Phosphopeptide Enrichment protocol: Priming Buffer and Syringe Wash Buffer

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 4, 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 5, 6, 8

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96-LP

5, 6, 8

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottoms

Agilent 201254-100 5, 6, 8

*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner and BioRad PCR plates are not compatible with the 25 L cartridges at deck locations 7 and 9.

Labware Manufacturer part number*

Deck location options

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Equilibration and Cartridge Wash Buffer Elution Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Phosphopeptide Enrichment

The Reagent Volume Calculator is a Microsoft Excel file that contains a Calculator worksheet. You enter the number of columns to process, whether to perform the Collect Flow Through options, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figure shows the Reagent Volume Calculator.

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Figure Phosphopeptide Enrichment Calculator worksheet

Preparing the buffers

CAUTION Make sure you filter salt-containing buffers if salt precipitation is a risk. Salt precipitates can clog cartridges and cause poor performance.

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the following buffers as a starting point for optimizing the AssayMAP phosphopeptide enrichment chemistry.

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Table Reagent preparation

Note: All suggested solutions listed as percentages are volume/volume formulations. Note: The source of NH3 for these buffers is aqueous ammonium hydroxide, which is commonly listed as %NH3 in H2O from commercial suppliers.

Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the reagents into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

Reagent (deck location)

Composition and comments

Priming Buffer & Syringe Wash Buffer (deck location 5)

This dual-purpose buffer serves as both the Priming Buffer and Syringe Wash Buffer during the protocol run. Do not alter the composition without evaluating how the change affects the priming step. Organic solvent in the buffer helps to purge entrained air within the cartridge resin bed during priming and is effective at removing hydrophobic species from syringes as part of the stringent syringe wash. Priming with a high-pH buffer helps to prepare TiO2 cartridges better than priming with a low-pH buffer. The following buffers have been used successfully with the indicated cartridges. Fe(III)-NTA: 99.9% ACN : 0.1% TFA TiO2 : 50% ACN : 45% H2O : 5% NH3

Elution Buffer (deck location 6)

Dilute solutions of aqueous ammonia are sufficient to elute phosphopeptides from Fe(III)-NTA and TiO2 cartridges. Recovery of hydrophobic peptides may be improved with the addition of small percentages of acetonitrile. The following buffers have been successfully used with the indicated cartridges. Fe(III)-NTA: 99 % H2O : 1% NH3 TiO2: 80 % H2O : 15% ACN : 5% NH3

Equilibration & Cartridge Wash Buffer (deck location 8)

This solution should be similar in composition to the sample loading buffer. The following buffers have been successfully used with the indicated cartridges. Fe(III)-NTA: 80% ACN : 19.9% H2O : 0.1% TFA TiO2: 50% ACN : 48 % H2O : 2% TFA

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IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and the cartridge positions in the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 434.

To dispense the solutions into the labware: 1 Optional. Label the labware so that you can easily identify them. 2 Add the specified volume of Priming & Syringe Wash Buffer into the labware to be

placed at deck location 5. 3 Add the specified volume of Elution Buffer into the labware to be placed at deck

location 6. 4 Add the specified volume of Equilibration & Cartridge Wash Buffer into the labware

to be placed at deck location 8. 5 If necessary, centrifuge the reagent labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare the samples immediately before running the Phosphopeptide Enrichment protocol, or keep the plates lidded until the run begins.

When preparing the samples, you must: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to optimize the binding conditions. Determine the volume of samples to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulates

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

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Adjusting the sample composition

Optimal conditions for phosphopeptide binding to AssayMAP phosphopeptide enrichment cartridges In a review of relevant literature for phosphopeptide enrichment using various titanium dioxide formats (228) and immobilized metal affinity chromatography (IMAC) formats (8, 2955), there was no clear consensus on sample and reagent formulations that yielded the optimum phosphopeptide enrichment ratio, the highest absolute phosphopeptide recovery, and the greatest reproducibility. In the majority of these studies, reagent formulations were optimized for the experiment at hand. Similarly, phosphopeptide enrichment using AssayMAP Fe(III)-NTA and TiO2 cartridges requires some degree of chemistry optimization and method development depending on the desired analytical result. No one set of chemistry conditions will be universally optimal. However, there are some chemistry trends that can serve as reasonable starting points for method development. Typically, samples for phosphopeptide enrichment using Fe(III)-NTA and TiO2 have first been subjected to a reversed-phase cleanup (desalting) step. Samples are generally in a phosphate-free, TFA-containing, low-pH buffer (pH 2.4 to 2.8) with some amount of organic solvent. Low-pH conditions reduce non-specific binding of non- phosphopeptides by facilitating protonation of acidic side-chains and C-termini of peptides, whereas the phosphoryl groups of phosphopeptides remain negatively charged at low pH to promote binding in a combination of ion exchange and metal coordination to the stationary phase (2, 56). The presence of organic solvent (typically 25% to 80% acetonitrile) in the sample and wash solutions helps to reduce nonspecific binding of non-phosphopeptides. Organic solvent serves to modulate the pKa values of ionizable species and the effect can be very different depending on the chemical species (57, 58). Achieving high phosphopeptide enrichment often means finding conditions that maximize the difference in the pKa values of phosphoryl and carboxyl groups such that under a given set of solvent conditions, the phosphoryl groups are negatively charged while carboxyl groups are neutral (45, 49). This is accomplished by adjusting the type and concentration of both the acid modifier and organic solvent present in the samples and wash buffers. Specificity can also be improved for phosphopeptide enrichment by TiO2 with the addition of small-molecule organic acids (such as, glycolic acid, lactic acid, and dihydroxybenzoic acid), which may further reduce nonspecific binding of non-phosphopeptides by competitively displacing weakly bound acidic, non-phosphopeptides.

Sample components that cause concerns Samples should be free of phosphates, free of particulates, and contain no or low concentrations of salts, denaturants, and chaotropes. The best results are typically achieved using samples first subjected to reversed-phase cleanup (desalting) before phosphopeptide enrichment. Solubilization of peptide samples after desalting and drying can be problematic when using high levels of organic solvent in the sample solubilization or reconstitution buffer. Solubility is often promoted by first adding the aqueous component of the buffer to the sample along with thorough mixing. This is followed by a slow titration of the organic portion of the buffer into the sample until the desired aqueous or organic ratio is achieved. Poor enrichment efficiency can be caused by insoluble or precipitated peptides that can form particulates large enough to collect at the top of the resin bed in the cartridge during sample loading. These particulates will mostly likely contain a very high percentage of non-phosphopeptides. If the wash buffer is insufficient to solubilize the particulates collected at the top of the cartridge, it is likely that the high-pH, aqueous

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conditions used for elution will solubilize the particulates. The peptides from the particulates will co-elute with the phosphopeptides and decrease the percent enrichment. If sample particulates form post filtration, attempt to refilter the sample or pellet the particulates by centrifugation before sample loading.

Sample load volume and cartridge capacity

The AssayMAP Phosphopeptide Enrichment protocol permits loading of up to 1000-L sample volumes onto AssayMAP phosphopeptide enrichment cartridges. For sample volumes > 250 L, the protocol will iteratively load samples onto cartridges to stay within the maximum AssayMAP syringe volume (250 L). For each AssayMAP Fe(III)-NTA and TiO2 cartridge, the quantitative binding capacity ( 90% recovery) depends on the chemical characteristics of the phosphopeptide targets and the sample matrix. Matrix effects include the overall sample complexity and the presence and abundance of certain of solvents, buffers components, additives, and the pH of the sample. For a tryptic digest of bovine a-casein, ~10 mole phosphate per mole protein (59), quantitative recovery of phosphopeptides is achieved with up to 1200-g digest (~500 nmol phosphate) loaded onto TiO2 cartridges and up to 150-g digest (~ 65 nmol phosphate) loaded onto Fe(III)-NTA cartridges. AssayMAP Fe(III)-NTA cartridges have an iron content greater than 100 nmol Fe(III) per cartridge as determined by inductively coupled plasmaoptical emission spectroscopy (ICP-OES). For enrichment of the small molecule test substrate phenylphosphate (4, 27), the cartridge capacity at saturation is approximately: 115 g (~ 660 nmol) per TiO2 cartridge

16 g (~ 92 nmol) per Fe(III)-NTA cartridge

Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must also match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the figure below shows.

The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based upon the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use the AssayMAP Resin- Free cartridges to fill the empty column and row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash step.

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Figure Example of sample microplate and reservoir layout: A) Multiple of 8 samples, B) Not a multiple of 8

See Labware on page 429 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

An excess (overage) volume ensures that a microplate well does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. See Using the Reagent Volume Calculator for Phosphopeptide Enrichment on page 431. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

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Running the protocol

The Phosphopeptide Enrichment protocol: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the peptides from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the cartridge seating station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the reagents. See Preparing the solutions on page 430. Prepare the samples. See Preparing the samples on page 434.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See the System Startup/Shutdown v3.0 User Guide utility.

Transfer the cartridges to the Cartridge & Tip Seating Station. See Cartridge Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the cartridge bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent Technologies does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 428.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Phosphopeptide Enrichment application.

To set up the protocol: 1 Open the App library. 2 Locate Phosphopeptide Enrichment, and then click App.

The Phosphopeptide Enrichment application opens.

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the protocol run on page 444. To modify the method or create a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

9 To save the method:

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a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 446.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 5) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

Equilibrate Aspirates Equilibration Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates samples (deck location 4) into the syringes, and then dispenses them through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 7).

5 L: 100 5 3

25 L: 100 5 3

Range: 01000 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the sample flow-through in the Organic Waste plate (deck location 3).

Cup Wash Rinses the cartridge cups with Cartridge Wash Buffer (deck location 8), and then discards the liquid into the Organic Waste plate (deck location 3).

5 L: 25 3

25 L: 25 3

Range: 0100 010

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Internal Cartridge Wash

Aspirates Cartridge Wash Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 7).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash flow-through at the Flow Through Collection (deck location 7). If not selected, discards the Internal Cartridge Wash flow- through into the Organic Waste (deck location 3).

Stringent Syringe Wash

Aspirates Syringe Wash Buffer (deck location 5) into the syringes, and then dispenses it into the Organic Waste plate (deck location 3).

5 L: 50 2

25 L: 50 2

Range: 0250 010

Elute Aspirates Elution Buffer (deck location 6) into the syringes, and then dispenses it through the cartridges into the Eluate Collection plate (deck location 9).

5 L: 25 5 1

25 L: 125 5 1

Range: 0250 0.1500 010

Eluate Discard

If selected, a specified initial volume of the eluate is discarded into the Organic Waste plate (deck location 3), or collected in the Flow Through Collection plate (deck location 7).

5 L: 0

25 L: 0

Range: 0250

Add to Flow Through

If selected, collects the Eluate Discard in the Flow Through Collection plate (deck location 7). If not selected, discards the Eluate Discard into the Organic Waste plate (deck location 3).

Existing Collection Volume

Specifies the volume of liquid present in the Eluate Collection plate (deck location 9) at the beginning of the run.

5 L: 0

25 L: 0

Range: 0300

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see the Protocol stepwise guidelines on page 447. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 458.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner and BioRad PCR plates are not compatible with the 25 L cartridges at deck locations 7 and 9.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

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After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol. The default protocol with 100-L samples and 5-L/min sample loading flow rate should take approximately 50 minutes to complete.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

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Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Phosphopeptide Enrichment

protocol to your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

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Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply

too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

Initial Syringe Wash This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

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Prime This step removes entrained air from the resin bed and properly wets the surface of the resin. In preparation for priming, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. For the most effective priming of AssayMAP Fe(III)-NTA and TiO2 cartridges, the Priming Buffer requires that the solution contain at least 50% organic solvent. Higher concentrations of organic solvent are also acceptable. This step is selected by default. Volume (L). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default value diminishes the ability to effectively remove entrained air from the resin bed. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. Note: The Equilibration Buffer is also used for the Cartridge Wash Buffer. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes, and then performs an external syringe wash at the wash station to remove any sample remaining on the outside of the probes before mounting the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or the Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. The protocol accommodates sample volumes up to 1000 L to be dispensed through the AssayMAP phosphopeptide enrichment cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L. Each syringe has a maximum capacity of 250 L. When sample volumes are greater than 250 L, the protocol will iteratively load samples through the cartridges. To determine the number and volume of iterative load steps, the protocol uses the following formulas: # of times to load = total sample volume/250,

where # times to load is rounded up to nearest integer volume of each load = sample volume/# of times to load For example, if the total sample volume is 900 L, then: # times to load = 900/250 = 3.6, which is rounded up to 4 volume of each load = 900/4 = 225 If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

IMPORTANT Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For more information, see Preparing the sample plates on page 436.

To determine the volume of sample to load, see Sample load volume and cartridge capacity on page 436. This step is selected by default.

Protocol step Guidelines and notes

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Volume (L). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. Large sample volumes (> 250 L) may require slightly more excess sample due to evaporation. Default: 100 Practical: 101000 Range: 01000 Note: The lower the sample volume the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than 5 L/min may not significantly increase analyte binding. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed into the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step is dispensed into the Organic Waste plate. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.

Protocol step Guidelines and notes

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Cup Wash This step removes the residual sample solution that may remain above the resin bed after the Load Samples step. The Cup Wash step aspirates Cartridge Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Organic Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

Internal Cartridge Wash

This step uses Cartridge Wash Buffer to wash non-specifically bound molecules from the resin bed. In preparation for the Internal Cartridge Wash, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Cartridge Wash Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates Cartridge Wash Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or the Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. If the Load Samples step is selected, the first 5 L (5 L cartridges) or 25 L (25 L cartridges) of Cartridge Wash Buffer 1 is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash volume minus the chase volume is dispensed at the Internal Cartridge Wash flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. This step is selected by default.

Protocol step Guidelines and notes

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Internal Cartridge Wash (continued)

Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increase the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the flow-through from Internal Cartridge Wash step is dispensed in the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from Internal Cartridge Wash is dispensed into the Organic Waste plate. This step is not selected by default.

Protocol step Guidelines and notes

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Stringent Syringe Wash

This step cleans the syringes with the Syringe Wash Buffer prior to elution. The Stringent Syringe Wash step aspirates the Syringe Wash Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the Organic Waste plate. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

Elute This step uses Elution Buffer to elute bound analytes from the cartridges. In preparation for elution, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the eluate is mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 L, the samples will not be mixed. You can also select the Eluate Discard and Add to Flow Through substeps, which are described in the following rows of this table. This step is selected by default.

Protocol step Guidelines and notes

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Elute (continued) Volume (L). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges, or 5075 L for 25 L cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Note: The Eluate Collection plate must be able to accommodate the total volume, which is determined by summing the net elution volume (Elute volume - Eluate Discard volume) and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Volume for 5 L cartridges:

Default: 25 Practical: 1030 Range: 0250

Volume for 25 L cartridges: Default: 125 Practical: 50150 Range: 0250

Note: Setting the volume to zero skips all Elute tasks except syringe washing. Flow rate (L/min). A flow rate slower than 5 L/min is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Eluate Discard This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. If the Eluate Discard step is selected, the specified volume is dispensed into the Organic Waste plate or Flow Through Collection plate (if the Add to Flow Through step is selected). The remaining Elution Buffer is dispensed through cartridges into the Eluate Collection plate.

Example: If the Elute, Eluate Discard, and Add to Flow Through steps are all selected with the following settings: Elute volume = 15 L (5 L cartridges) or 40 L (25 L cartridges) Eluate Discard volume = 2 L (5 L cartridges) or 10 L (25 L cartridges) the first 2 L (5 L cartridges) or 10 L (25 L cartridges) eluate from the cartridges will be discarded into the Flow Through Collection plate, and the remaining 13 L (5 L cartridges) or 30 L (25 L cartridges) eluate will be collected in the Eluate Collection plate.

Select the Eluate Discard step in situations where minimizing the volume of eluate is important. For AssayMAP cartridges, the initial elution volume (~2 L for 5 L cartridges and ~10 L for the 25 L cartridges) contains small or no measurable amounts of analyte. This step is not selected by default. Volume (L). The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. Default: 0 Practical:

5 L cartridges: 02 25 L cartridges: 010

Range: 0250

Add to Flow Through

If selected, this step dispenses the Eluate Discard volume into the Flow Through Collection plate. If the Add to Flow Through step is not selected, the Eluate Discard is dispensed into the Organic Waste plate. This step is not selected by default. The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected. Note: The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected.

Protocol step Guidelines and notes

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Existing Collection Volume

This step enables you to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. The Existing Collection Volume and the net volume from the Elute step (Elute volume - Eluate Discard volume) feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head into and out of the wells during elution and eluate mixing in a volume- dependent manner. For the maximum practical working volumes of labware for eluate collection, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, for adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (L): Default: 0 Practical: 0250 Range: 0300

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate. No solution is added into the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L of DI water into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. Note: In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Phosphopeptide Enrichment protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Starting protocol 2 Parks all cartridges that might have been loaded on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes.

Prime 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

5 Aspirates 10 L of Priming Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

5 Aspirates the Priming Buffer.

2 Mounts the cartridges onto the head.

3 Dispenses the Priming Buffer through the cartridges into the Organic Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

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Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

8 Aspirates 10 L of Equilibration Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Equilibration Buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Equilibration Buffer through the cartridges to equilibrate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Load Samples 4 Aspirates the samples into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

7 Dispenses the samples through the cartridges and into the Flow Through Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 8 Aspirates the Cartridge Wash Buffer into the syringes.

2 Washes the cartridge cups and exercises the cartridges off task.

3 Dispenses the Cartridge Wash buffer into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

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Internal Cartridge Wash

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the cartridge tips.

8 Aspirates 10 L of Cartridge Wash Buffer 1 for the cartridge air- gap-prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Cartridge Wash Buffer into the syringes for sample chase and the Internal Cartridge Wash steps.

2 Mounts the cartridges on the head.

3 Dispenses 5 L (5 L cartridges) or 25 L (25 L cartridges) Cartridge Wash Buffer through the cartridges at the Load Samples flow rate for the sample chase step.

3 Dispenses the remaining Cartridge Wash buffer through the cartridges at the Internal Cartridge Wash flow rate and into the Organic Waste plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Stringent Syringe Wash

5 Aspirates the Syringe Wash Buffer into the syringes.

3 Dispenses the Syringe Wash Buffer into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Elute 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

6 Aspirates 10 L of Elution Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Elution Buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates the Elution Buffer.

2 Mounts the cartridges on the head.

9 Elutes the samples into the Elution Collection Plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

9 Mixes the eluates.

1 Washes the syringes.

Final Syringe Wash 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

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Reference library

1 Russell, J.D., Murphy, S.M., Agilent AssayMAP Bravo Technology Enables Reproducible Automated Phosphopeptide Enrichment from Complex Mixtures Using High-Capacity Fe(III)-NTA Cartridges, Agilent Application Note 5991-6073EN, March 2016

2 Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P., & Jrgensen, T.J.D. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics, 2005, 4:873-886.

3 Klemm, C. et al. Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides. J. Mass Spectrom., 2006, 41:1623-1632.

4 Liang, S.S., Makamba, H., Huang, S.Y., & Chen, S.H. Nano-titanium dioxide composites for the enrichment of phosphopeptides. J. Chromatogr., 2006, 1116:38-45.

5 Olsen, J.V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell, 2006, 127:635-648.

6 Thingholm, T.E., Jorgensen, T.J.D., Jensen, O.N., & Larsen, M.R. Highly selective enrichment of phosphorylated peptides using titanium dioxide. Nat. Protocols, 2006, 1:1929-1935.

7 Hsieh, H.-C., Sheu, C., Shi, F.-K., & Li, D.-T. Development of a titanium dioxide nanoparticle pipette-tip for the selective enrichment of phosphorylated peptides. J. Chromatogr., 2007, 1165:128-135.

8 Jensen, S.S. & Larsen, M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun. Mass Spectrom., 2007, 21:3635-3645.

9 Mazanek, M. et al. Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis. Nat. Protocols, 2007, 2:1059-1069.

10 Yu, L.-R. et al. Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra. J. Proteome. Res., 2007, 6:4150-4162.

11 Macek, B. et al. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol. Cell. Proteomics, 2008, 7:299-307.

12 Simon, E.S., Young, M., Chan, A., Bao, Z.-Q., & Andrews, P.C. Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution. Anal. Biochem., 2008, 377:234-242.

13 Li, Q.-r., Ning, Z.-b., Tang, J.-s., Nie, S., & Zeng, R. Effect of peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity. J. Proteome. Res., 2009, 8:5375- 5381.

14 Oppermann, F.S. et al. Large-scale proteomics analysis of the human kinome. Mol. Cell. Proteomics, 2009, 8:1751-1764.

15 Thingholm, T. and Larsen, M. in Phospho-Proteomics, Vol. 527. (ed. M. Graauw), 57-66 (Humana Press, 2009).

16 Yu, Y.-Q., Fournier, J., Gilar, M., & Gebler, J.C. Phosphopeptide enrichment using microscale titanium dioxide solid phase extraction. J. Sep. Sci., 2009, 32:1189- 1199.

17 Aryal, U.K. & Ross, A.R.S. Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry. Rapid Commun. Mass Spectrom., 2010, 24:219-231.

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18 Eriksson, A. et al. Optimized protocol for on-target phosphopeptide enrichment prior to matrix-assisted laser desorption-ionization mass spectrometry using mesoporous titanium dioxide. Anal. Chem., 2010, 82:4577-4583.

19 Gates, M., Tomer, K. & Deterding, L. Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses. J. Am. Soc. Mass Spectrom., 2010, 21:1649-1659.

20 Iwase, Y. et al. A fully automated phosphopeptide purification system for large- scale phosphoproteome analysis. J. Biochem., 2010, 147:689-696.

21 Engholm-Keller, K., Hansen, T.A., Palmisano, G. & Larsen, M.R. Multidimensional strategy for sensitive phosphoproteomics incorporating protein prefractionation combined with SIMAC, HILIC, and TiO2 chromatography applied to proximal EGF signaling. J. Proteome. Res., 2011, 10:5383-5397.

22 Engholm-Keller, K. & Larsen, M.R. Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds - Applications in acidic modification-specific proteomics. Journal of Proteomics, 2011, 75:317-328.

23 Kettenbach, A.N. & Gerber, S.A. Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments. Anal. Chem., 2011, 83:7635-7644.

24 Pinkse, M.W., Lemeer, S., & Heck, A.J. A protocol on the use of titanium dioxide chromatography for phosphoproteomics. Methods Mol. Biol., 2011, 753:215-228.

25 Fukuda, I. et al. Optimization of enrichment conditions on TiO2 chromatography using glycerol as an additive reagent for effective phosphoproteomic analysis. J. Proteome. Res., 2013.

26 Lajoie, M.J. et al. Genomically recoded organisms expand biological functions. Science, 2013, 342:357-360.

27 Richardson, B.M., Soderblom, E.J., Thompson, J.W., & Moseley, M.A. Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics. J Biomol Tech, 2013, 24:8-16.

28 Yu, L.R. & Veenstra, T. Phosphopeptide enrichment using offline titanium dioxide columns for phosphoproteomics. Methods Mol. Biol., 2013, 1002:93-103.

29 Li, S. & Dass, C. Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides. Anal. Biochem., 1999, 270:9-14.

30 Posewitz, M.C. & Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem., 1999, 71:2883-2892.

31 Hart, S.R., Waterfield, M.D., Burlingame, A.L., & Cramer, R. Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS. J. Am. Soc. Mass Spectrom., 2002, 13:1042-1051.

32 Ficarro, S. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol., 2002, 20:301 - 305.

33 Stensballe, A. & Jensen, O.N. Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides. Rapid Commun. Mass Spectrom., 2004, 18:1721-1730.

34 Barnouin, K.N. et al. Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol. Proteomics, 2005, 5:4376-4388.

35 Corthals, G.L., Aebersold, R. & Goodlett, D.R. in Methods in Enzymology, Vol. Volume 405. (ed. A.L. Burlingame), 66-81 (Academic Press, 2005).

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36 Kange, R. et al. Comparison of different IMAC techniques used for enrichment of phosphorylated peptides. Journal of Biomolecular Techniques, 2005, 16:91-103.

37 Seeley, E.H., Riggs, L.D. & Regnier, F.E. Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme. J. Chromatogr. B., 2005, 817:81-88.

38 Ndassa, Y.M., Orsi, C., Marto, J.A., Chen, S., & Ross, M.M. Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications. J. Proteome. Res., 2006, 5:2789-2799.

39 Feng, S. et al. Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis. Proteomics, 2007, 7:351-360.

40 Feng, S. et al. Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis. Mol. Cell. Proteomics, 2007, 6:1656-1665.

41 Imanishi, S.Y., Kochin, V., & Eriksson, J.E. Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography. Proteomics, 2007, 7:174- 176.

42 Lee, J. et al. Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS. Mol. Cell. Proteomics, 2007, 6:669-676.

43 Steen, H., Stensballe, A. & Jensen, O.N. Phosphopeptide purification by IMAC with Fe(III) and Ga(III). Cold Spring Harbor Protocols, 2007, 2007:pdb.prot4607.

44 Thingholm, T.E., Jensen, O.N., Robinson, P.J., & Larsen, M.R. SIMAC (Sequential Elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. Mol. Cell. Proteomics, 2008, 7:661-671.

45 Tsai, C.-F. et al. Immobilized metal affinity chromatography revisited: pH/Acid control toward high selectivity in phosphoproteomics. J. Proteome. Res., 2008, 7:4058-4069.

46 Villen, J. & Gygi, S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nature Protocols, 2008, 3:1630- 1638.

47 Ficarro, S.B. et al. Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment. Anal. Chem., 2009, 81:4566-4575.

48 Swaney, D.L., Wenger, C.D., Thomson, J.A., & Coon, J.J. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. U. S. A., 2009, 106:995-1000.

49 Ye, J. et al. Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples. J. Proteome. Res., 2010, 9:3561-3573.

50 El Idrissi, K., Eddarir, S., Tokarski, C., & Rolando, C. Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: Comparison between polymer brush coating and surface functionalization. J. Chromatogr. B., 2011, 879:2852-2859.

51 Dephoure, N. & Gygi, S.P. A solid phase extraction-based platform for rapid phosphoproteomic analysis. Methods, 2011, 54:379-386.

52 Frantzi, M. et al. IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers. J. Proteome. Res., 2013.

53 Mertins, P. et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Meth, 2013, advance online publication.

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54 Zhu, L., Zhang, J. & Guo, Y. Enhanced detection and desalting free protocol for phosphopeptides eluted from immobilized Fe (III) affinity chromatography in direct MALDI TOF analysis. Journal of Proteomics, 2014, 96:360-365.

55 Ruprecht, B. et al. Comprehensive and reproducible phosphopeptide enrichment using Fe-IMAC columns. Mol. Cell. Proteomics, 2014.

56 Holmes, L.D. & Schiller, M.R. Immobilized Iron(III) metal affinity chromatography for the separation of phosphorylated macromolecules: Ligands and applications. J. Liq. Chromatogr. Rel. Technol., 1997, 20:123-142.

57 Subirats, X., Ross, M. & Bosch, E. On the effect of organic solvent composition on the pH of buffered HPLC mobile phases and the pKa of analytes - A review. Separation & Purification Reviews, 2007, 36:231-255.

58 Bosch, E., Espinosa, S. & Ross, M. Retention of ionizable compounds on high- performance liquid chromatography: III. Variation of pK values of acids and pH values of buffers in acetonitrile-water mobile phases. J. Chromatogr., 1998, 824:137-146.

59 Modler, H.W. Functional properties of nonfat dairy ingredients - a review. Modification of products containing casein1,2. J. Dairy Sci.,68:2195-2205.

See the Agilent AssayMAP Bravo Citation Index for published papers that used the AssayMAP Bravo Platform.

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This chapter contains the following topics: App description on page 468 Before you start on page 468 Preparing the solutions on page 472 Preparing the samples on page 476 Running the protocol on page 479 Assay development guidelines and protocol notes on page 488 Reference library on page 504

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App description

Protein Cleanup v3.0. This application enables automated cleanup of from 1 to 96 protein samples in a single run.

Before you start

This topic lists the required hardware, software, AssayMAP cartridges, labware, and reagents for running the Protein Cleanup protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Optional equipment. You might need the following when preparing the samples and reagents:

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate vacuum concentrator for concentration and drying of samples Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP cartridges

The following table lists the available AssayMAP cartridges for performing Protein Cleanup on the AssayMAP Bravo Platform. Each cartridge type can be purchased as a rack of 96 cartridges.

For more details about the available cartridges, see the Agilent AssayMAP Bravo Cartridges Selection Guide or the AssayMAP Cartridges page on Agilent.com.

Cartridge use and storage guidelines

See the cartridge box label for storage guidelines. Follow these guidelines to get the best performance from AssayMAP cartridges: Use only primed and equilibrated cartridges.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Cartridge type Agilent part number

5 L cartridge 25 L cartridge

AssayMAP Reversed-Phase (RP-W) cartridge rack G5496-60086 G5496-60024

AssayMAP Resin-Free cartridge rack This cartridge can be used for mock runs or as cartridge placeholders if only partial columns of RP-W 5- or 25-L cartridges are required. For more information, see Preparing the samples on page 476.

G5496-60009

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IMPORTANT Cartridges ship dry and, therefore, contain air entrained in the resin bed. Failure to prime the cartridges can prevent the reagents and buffers from accessing parts of the resin bed, resulting in reduced capacity and poor reproducibility.

Do not allow wetted cartridges to dry out. Note: Cartridges will not dry out during the course of a normal application run. Cartridges can dry out if they are exposed to air for extended periods (e.g., >1 hour) after they have been primed and equilibrated. If you need to store primed and equilibrated cartridges for a short period, ensure that you use the lidded blue rack-receiver plate stack with an appropriate solution in the receiver plate chimneys such that the cartridge tips are submerged in the solution.

AssayMAP cartridges are intended to be single-use consumables. Agilent does not provide a performance guarantee for cartridges that have been used more than once.

AssayMAP RP-W cartridges tolerate brief exposure to solutions with pH values of approximately 114.

Labware

Labware requirements vary depending on experimental design. The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware Manufacturer part number* Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3, 58

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 3, 58

96 Agilent, 2mL Square Deep Well labware Agilent 204353-100 3, 7

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Reagents

The volume, type, and concentration of reagents required for protein cleanup vary depending on sample characteristics and the desired analytical result. Consult the published scientific literature and Reference library on page 504 to find specific chemistry examples. See the Agilent AssayMAP Bravo Citation Index for published papers that use the AssayMAP Protein Cleanup application. By default, the syringes are rinsed thoroughly with deionized water at the wash station after completing the protocol to reduce the risk of premature syringe failure. To perform more stringent syringe washing between runs, use the Syringe Wash utility. For details, see the Syringe Wash v3.0 User Guide. All labware require volume overage for the protocol to execute properly. Use the Reagent Volume Calculator to determine volume requirements for specific protocol conditions. See Preparing the solutions on page 472.

Preparing the solutions

The following solutions are required for the Protein Cleanup protocol: Priming & Syringe Wash Buffer Equilibration & Cartridge Wash Buffer

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 38

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7, 9

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7, 9**

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 39

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 39

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 39

96 Costar 3363, PP Conical Bottom Corning Costar 3363 39

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 4, 7, 9

96 V11 Manual Fill Reservoir Agilent G5498B#049 5, 6, 8

*For dimensionally equivalent alternatives and other labware details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **The Greiner PCR plate is not compatible with the 25 L cartridges at deck locations 7 and 9.

Labware Manufacturer part number* Deck location options

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Elution Buffer

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Using the Reagent Volume Calculator for Protein Cleanup

The Reagent Volume Calculator is a Microsoft Excel file that contains the following: Calculator worksheet. You enter the number of columns to process, whether to

perform the Collect Flow Through option, the volume for each step in the protocol, the number of wash cycles to conduct, and the labware selection for each deck location. The calculator determines the volumes required based on your input, taking into consideration pipetting overage and evaporation concerns. Note: The pipetting overage suggested is generally conservative. The minimal overage may be greater or less depending on the volatility of the solution, the length of the run, and when the step occurs during the run. The overage volume can be optimized to minimize loss of precious reagents.

Reagent Recipe worksheet. You enter the concentrations of each component in your reagent, and the worksheet calculates the recipe volumes required.

To use the Reagent Volume Calculator: 1 Open the App Library. 2 Locate the application, and then click the corresponding Calculator button.

Microsoft Excel starts and displays the calculator. 3 Ensure that you enable content in Microsoft Excel. 4 Click one of the following:

Set defaults for 5L cartridges. Sets the values in the calculator using the values from the default method for the 5 L cartridges.

Set defaults for 25L cartridges. Sets the values in the calculator using the values from the default method for the 25 L cartridges.

5 Modify the values in the green boxes as required to match your specific method. As you change the values in the green boxes, the calculated values change. Note: The green box should remain green after you enter a value. If you enter a value that is outside the normal working range, the box becomes yellow. If you enter a value that is outside of the acceptable range, the box becomes red. To display the corresponding tooltip for a setting, mouse over a box that has a red triangle in the upper right corner.

The following figures show the worksheets of the Reagent Volume Calculator.

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Figure Protein Cleanup Calculator worksheet

Figure Protein Cleanup Reagent Recipe worksheet

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Preparing the solutions

The following table describes the reagents and deck locations. The AssayMAP protocols are blind to the composition of the solutions, so you can easily adapt your optimized chemistry. Agilent recommends the buffers listed in the following table as a starting point for optimizing the AssayMAP Protein Cleanup chemistry.

Table Reagent preparation

Dispensing the solutions

IMPORTANT To prevent evaporation, dispense the solutions into the labware immediately before running the protocol, or keep the plates lidded until the run begins.

IMPORTANT If you are using fewer than 96 cartridges, make sure you fill the labware to correspond with the sample layout in the sample plate and the cartridge positions in the 96AM Cartridge & Tip Seating Station. For more information, see Preparing the samples on page 476.

To dispense the solutions into the labware: 1 Optional. Label each piece of labware so that you can easily identify them.

Reagent (deck location) Composition and comments

Priming & Syringe Wash Buffer (deck location 5)

Typically 50% or greater organic (must be >25% organic), acidic, and identical in composition to the Elution Buffer to simplify the solution preparation, for example, 60% ACN: 39.9% H2O : 0.1% TFA

The high-percentage organic composition is essential for reversed-phase cartridges as they ship dry and must be wetted with a high percentage organic solution or they will have very little binding capacity. This solution, the other solutions used in this application, and the sample are typically acidic. Other more mass-spec-friendly acids can substitute for TFA.

Elution Buffer (deck location 6)

High percentage organic and acidic solution, for example, 60% ACN: 39.9% H2O: 0.1% TFA

Note: Carefully determine the percentage of organic solution and the pH as they both strongly influence the elution efficiency.

Equilibration & Cartridge Wash Buffer (deck location 8)

Very low or no organic and acidic solution. Typically, similar in the percentage organic and acidity to the sample, for example, 99.9% H2O : 0.1% TFA

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2 Add the specified volume of Priming & Syringe Wash Buffer into the labware to be placed at deck location 5.

3 Add the specified volume of Elution Buffer into the labware to be placed at deck location 6.

4 Add the specified volume of Equilibration & Cartridge Wash Buffer into the labware to be placed at deck location 8.

5 If necessary, centrifuge the filled labware to remove bubbles. Note: You can use the Reagent Aliquot utility to dispense the buffers. For details, see Reagent Aliquot v2.0 User Guide on page 518.

Preparing the samples

IMPORTANT To minimize evaporation, prepare samples immediately before running the Protein Cleanup protocol, or keep the plates lidded until the run begins.

When preparing the samples: Remove macromolecular particulates before the samples are loaded onto

AssayMAP cartridges. Adjust the buffer composition to the optimal binding conditions (for example, low

pH and low organic). Determine the sample volume to load on the AssayMAP cartridges. Transfer the samples to the microplate you want to use for the protocol run.

Removing macromolecular particulate

Make sure the samples are free of macromolecular particulates, such as large protein aggregates and cellular debris to prevent clogging the cartridges. Samples should be filtered through a 0.45-m filter or centrifuged at a high g-force immediately before loading on an AssayMAP cartridge.

Adjusting the buffer composition

The Protein Cleanup application can be used for a wide variety of proteins. Given the range of physiochemical properties that proteins display, you should examine the relevant scientific literature to determine the sample buffer conditions that favor efficient binding and avoid negatively affecting your protein of interest. In general, acidification, which can be accomplished by the addition of TFA, formic acid, or acetic acid to the sample, improves binding to reversed phase resins such as RP-W.

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Agilent app note 5991-6478EN (Reference library on page 504) provides a detailed analysis of the optimal conditions for using the RP-W cartridges for the cleanup of denatured antibodies. This app note focuses on the cleanup of denatured antibodies to enable rapid trypsin digestion, but it also provides a general approach to optimize the conditions to clean up your protein of interest. Avoid samples containing organic solvents or some types of detergents as they might inhibit binding to the cartridge. For example, loading samples in a buffer containing greater than 5% acetonitrile might inhibit binding of some proteins. If you have concerns about a specific buffer component, you should survey the scientific literature for any known effects of this type of molecule on reversed-phase resins. The Reagent Transfer utility is a simple way to adjust the pH or dilute your sample before loading it onto the RP-W cartridge. For instructions, see Reagent Transfer v3.0 User Guide on page 525. Note: A reverse-phase protein clean up may not work for all proteins due to limited solubility in the high organic solvent, which is required for elution. In addition, when desalting a denatured protein, the removal of the denaturation reagent may cause solubility problems.

Determining the volume of sample to load

The Protein Cleanup protocol permits loading of up to 1000-L sample volume onto the AssayMAP cartridges. If the sample volume is greater than 250 L, the protocol will iteratively load samples onto the cartridges to stay within the maximum AssayMAP syringe volume (250 L).

What is the binding capacity of the cartridge? Two ways to express the binding capacity of a cartridge are quantitative binding capacity and total binding capacity: Quantitative binding capacity. The maximum mass of protein that can bind to the

cartridge in a single pass, where less than 10% of the load appears in the flow- through. The quantitative binding capacity depends on the composition of the solution in which the protein is bound. For denatured antibodies, the binding capacity is slightly reduced when there is greater than 1.5 M guanidine in the sample and if the sample is not acidified.

Total binding capacity. The maximum mass of protein that can bind to the cartridge. This can only be achieved only by loading significantly more protein than can be bound by the cartridge. This value is significantly greater than the quantitative binding capacity.

See the Agilent AssayMAP Bravo Cartridges Selection Guide for detailed information about the binding capacity for the RP-W cartridges.

What is the concentration of the target in the sample? If you know the approximate concentration of the target molecule in your sample and you are working within the quantitative binding range of the cartridge, you can determine the volume of sample to load as follows:

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Preparing the sample plates

Planning the microplate setup Before transferring the samples, you should plan the layout of the samples in the microplate. Consider the following: You can process 1 to 96 samples in parallel. The position of the samples in the

microplate dictates the positions of the cartridges in the 96AM Cartridge & Tip Seating Station. These positions must match the locations of the buffer solutions in microplates and reservoirs.

If you have fewer than 96 samples, make sure the samples occupy full columns in the microplate, as the following figure shows.

The default protocol settings assume that samples will be arranged in multiples of 8 in a column-based configuration. Also, the AssayMAP Bravo Platform applies differential pressure to seat cartridges based upon the number of full columns of cartridges. To achieve proper cartridge seating, entire columns must be used.

If the number of samples you have is not a multiple of 8, use the AssayMAP Resin- Free cartridges to fill the empty column and row positions. This will prevent liquids from dripping on the deck or being dispensed on the deck during the Cup Wash step.

Figure Example of sample microplate and reservoir layout: A) Multiple of 8 samples, B) Not a multiple of 8

See Labware on page 471 for acceptable labware at each deck location.

Transferring the samples to the microplate

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

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An excess (overage) volume ensures that a microplate well or column does not fully deplete, which would result in aspiration of air into the syringes and then into the cartridges, compromising performance. The Reagent Volume Calculator shows the recommended overage for the labware types being used and automatically includes recommended overages in the volume it recommends per well. See Using the Reagent Volume Calculator for Protein Cleanup on page 473. Labware-specific overage recommendations are also presented in the Labware Reference Guide, which you can find in the Literature Library page of the Protein Sample Prep Workbench. More or less overage can be used depending on the volatility of the solution and the length of the run but the recommended overages are fine for most standard runs.

To transfer the samples to the microplate: 1 Run the Reagent Transfer utility or Reformatting utility to move samples into one of

the labware options for deck location 4. For instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623

2 If necessary, centrifuge the sample labware to remove bubbles.

Running the protocol

The Protein Cleanup protocol does the following: Washes the syringes. Primes and equilibrates the cartridges to prepare for sample loading. Loads the samples onto the cartridges. Removes non-specific binding molecules from the cartridges. Elutes the protein from the cartridges. For some of these operations the cartridges are mounted on the syringe probes, while for other operations the cartridges are parked in the cartridge seating station.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded.

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To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the buffers. See Preparing the solutions on page 472. Prepare the samples. See Preparing the samples on page 476. If applicable, make sure that you know which experiment ID to use to record the

steps executed during the utility and app runs. Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See

System Startup/Shutdown v3.0 User Guide. Transfer the cartridges to the 96AM Cartridge & Tip Seating Station. See Cartridge

Transfer v2.0 User Guide on page 506.

IMPORTANT Cartridges ship dry and therefore contain air entrained in the resin bed. Failure to prime the cartridges can prevent the sample and buffers from accessing parts of the bed, resulting in reduced capacity and poor reproducibility.

IMPORTANT Do not allow wetted cartridges to dry out. Agilent Technologies does not guarantee performance of stored cartridges following equilibration. See Cartridge use and storage guidelines on page 470.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Protein Cleanup application.

To set up the protocol: 1 Open the App Library. 2 Locate Protein Cleanup v3.0, and then click App.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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The Protein Cleanup application opens.

3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the protocol run on page 485.

To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the cartridge settings:

a Select the cartridge size from the list: 5 L Cartridges 25 L Cartridges

b In the box, type the number of full columns of cartridges to be used. The position of the columns of cartridges in the tip seating station must match the positions of the samples and solutions in the plates on the deck. Range: 112 Default: 1

CAUTION If the column selection is greater than the actual number of columns used, the Bravo Platform will apply too much force when mounting the cartridges, which can cause damage to both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required. To prevent potential equipment damage, ensure that the column selection is correct.

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CAUTION If the column selection in the software is less than the actual number of cartridges used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable. To obtain expected instrument performance, ensure that the column selection is correct.

IMPORTANT Each full column must contain eight cartridges. If a column contains fewer than eight packed cartridges, use the AssayMAP Resin-Free cartridges to fill the empty column positions.

7 Under Application Settings, select the check boxes of the steps that you want to perform, and enter the values for the selected steps. Note: For any unselected steps, ensure that the volume, flow rate, and wash cycles boxes are blank to avoid potential confusion when a experimental report is generated.

8 In the Labware Table area, select the labware you are using for the protocol run. Note: If all the steps that use a certain labware location are unchecked, ensure that the labware selection is No labware to avoid confusion when setting up the deck and when generating an experimental report. The Reagent volume calculator is a good resource for this decision because it returns a value of zero in the Volume per well required cell if no labware is needed.

9 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. Note: Agilent recommends that you use the cartridge size (5 L or 25 L) as a prefix to the name.

VWorks Plus. You must save the method before you can run it.

Application Settings

The following table gives a brief description of each setting. For details, including the full and practical ranges of values for a given setting, see the Assay development guidelines and protocol notes on page 488.

Table Application Settings overview

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

Initial Syringe Wash

Washes syringes at the wash station (deck location 1).

5 L: 3

25 L: 3

Range: 010

Prime Aspirates Priming Buffer (deck location 5) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 100 300 1

25 L: 250 300 1

Range: 0250 0.5500 010

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Equilibrate Aspirates Equilibration Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3).

5 L: 50 10 1

25 L: 250 10 1

Range: 0250 0.5500 010

Load Samples

Aspirates samples (deck location 4) into the syringes, and then dispenses them through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 7).

5 L: 100 5 3

25 L: 100 5 3

Range: 01000 0.1500 010

Collect Flow Through

If selected, collects the sample flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the sample flow-through into the Organic Waste plate (deck location 3).

Cup Wash Rinses the cartridge cups with Cartridge Wash Buffer (deck location 8), and then discards the liquid into the Organic Waste plate (deck location 3).

5 L: 25 3

25 L: 25 3

Range: 0100 010

Internal Cartridge Wash

Aspirates the Cartridge Wash Buffer (deck location 8) into the syringes, and then dispenses it through the cartridges into the Organic Waste plate (deck location 3) or into the Flow Through Collection plate (deck location 7).

5 L: 50 10 3

25 L: 250 10 3

Range: 0250 0.5500 010

Collect Flow Through

If selected, collects the Internal Cartridge Wash flow-through in the Flow Through Collection plate (deck location 7). If not selected, discards the Internal Cartridge Wash flow-through into the Organic Waste plate (deck location 3).

Stringent Syringe Wash

Aspirates the Syringe Wash Buffer (deck location 5) into the syringes, and then dispenses it into the Organic Waste plate (deck location 3).

5 L: 50 2

25 L: 50 2

Range: 0250 010

Elute Aspirates the Elution Buffer (deck location 6) into the syringes, and then dispenses it through the cartridges into the Eluate Collection plate (deck location 9).

5 L: 25 5 1

25 L: 125 5 1

Range: 0250 0.1500 010

Eluate Discard

If selected, a specified initial volume of the eluate is discarded into the Organic Waste plate (deck location 3), or collected in the Flow Through Collection plate (deck location 7).

5 L: 0

25 L: 0

Range: 0250

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. To decrease the run time, you can increase the flow rates to 500 L/min, change the wash cycles to 1, and decrease the volumes. Use the AssayMAP Resin-Free cartridges instead of packed cartridges for mock runs.

IMPORTANT The protocol will display an error message if cartridges are missing.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Note: The Greiner PCR plates are not compatible with the 25 L cartridges at deck locations 7 and 9.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form.

Add to Flow Through

If selected, collects the Eluate Discard in the Flow Through Collection plate (deck location 7). If not selected, discards the Eluate Discard into the Organic Waste plate (deck location 3).

Existing Collection Volume

Specifies the volume of liquid present in the Eluate Collection plate (deck location 9) at the beginning of the run.

5 L: 0

25 L: 0

Range: 0300

Final Syringe Wash

Washes the syringes at the wash station (deck location 1).

3

3

010

*For practical value ranges for the steps listed in this table and factors to consider when changing the default values, see Protocol stepwise guidelines on page 488. For a complete list of the robotic movements executed during a run, see Automation movements during the protocol on page 500.

Steps* Description Cartridge size

Volume (L)

Flow Rate (L/min)

Wash Cycles

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Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

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To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

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To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines and protocol notes

This topic explains the following: Each step of the protocol so that you can optimize the Protein Cleanup protocol to

your particular experimental design Automation movements during the protocol For details on how to use the Experiments Editor, see Using Experiment IDs on page 23.

Protocol stepwise guidelines

Protocol step Guidelines and notes

Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct. If the column selection is: Greater than the actual number of columns used, the Bravo Platform will apply

too much force when mounting the cartridges, which can damage both the cartridges and the AssayMAP syringes in the head. For example, if the software specifies 12 columns, but only 1 column of cartridges are in the seating station, the head will apply 12 times more force than what is required.

Less than the actual number of columns used, the Bravo Platform will not apply enough force to seat the cartridges properly. For example, if the software specifies 1 column, but 12 columns of cartridges are in the seating station, the head will apply 1/12th the force required to seat the cartridges properly. In this case, cartridges may fall off during the run or the volume of liquid that moves across the cartridge bed may be variable due to liquid moving past the syringe cartridges seal into the cartridge cup.

Default: 1 Range: 1-12

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Initial Syringe Wash This step removes any potential contaminants from the syringes at the wash station before the cartridges are mounted. During each Initial Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys and then moves by a fixed offset between the wash station chimneys to dispense to waste. This step is selected by default. Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Prime This step removes entrained air from the packed resin bed and properly wets the surface of the resin. In preparation for priming, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Priming Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. The AssayMAP reversed-phase cartridges (RP-W) used with the Protein Cleanup application require a Priming Buffer containing at least 25% organic solvent. As illustrated in Agilent app note 5991-6478EN (Reference library on page 504), reversed-phase cartridges (RP-W) that are primed with solutions containing a low percentage of organic solvent have greatly reduced binding capacity. Agilent recommends using a priming solution that contains at least 50% organic solvent, such as acetonitrile. This step is selected by default. Volume (L). The default volume is sufficient to wet and remove entrained air from the resin bed. Using less than the default volume may leave air in the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 100 Practical: 100250 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Prime tasks except syringe washing. Flow rate (L/min). A flow rate lower than the default value diminishes the ability to effectively remove entrained air from the cartridge. A flow rate faster than the default is not required and has not been tested. Default: 300 Practical: 300 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Equilibrate This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step. In preparation for equilibration, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Equilibration Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station. The Agilent reversed-phase cartridge (RP-W) typically used with the Protein Cleanup application require an equilibration solution that has a very low concentration of or no organic solvent for effective binding during the sample loading step. This step is selected by default. Volume (L). The default volume is equal to 10 column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time. Volume for 5 L cartridge:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridge: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Equilibrate tasks except syringe washing. Flow rate (L/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 L/min using the default volume may not equilibrate through the pores in the beads. Default: 10 Practical: 520 Range: 0.5500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Protocol step Guidelines and notes

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Load Samples This step allows the target analytes to bind to the surface chemistry of the resin bed. No liquid is removed or added to the cartridge cups before the sample loading begins. The assumption is that there is still liquid in the cups from the equilibration step that will prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. This step aspirates sample into the syringes, and then performs an external syringe wash at the wash station to remove any sample remaining on the outside of the probes before mounting the cartridges. The samples are dispensed through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any sample on the exterior of the cartridges, the cartridges are parked at the seating station, and the syringes are washed at the wash station. The protocol accommodates sample volumes up to 1000 L to be dispensed through the AssayMAP protein cleanup cartridges. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 L. Each syringe has a maximum capacity of 250 L. When sample volumes are greater than 250 L, the protocol will iteratively load volumes onto the cartridges. To determine the number and volume of iterative load steps, the protocol uses the following formulas: # of times to load = total sample volume/250,

where # times to load is rounded up to nearest integer volume of each load = sample volume/# of times to load For example, if the total sample volume is 900 L, then: # times to load = 900/250 = 3.6, which is rounded up to 4 volume of each load = 900/4 = 225 If Collect Flow Through is selected for the Load Samples step, be sure that the Flow Through Collection plate has sufficient maximum well capacity. For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

IMPORTANT Be sure to include the recommended labware-specific volume overage to prevent air from entering the cartridge. For more information, see Transferring the samples to the microplate on page 478.

This step is selected by default.

Protocol step Guidelines and notes

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Volume (L): The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. Large sample volumes (> 250 L) may require slightly more excess sample due to evaporation. Default: 100 Practical: 101000 Range: 01000 Note: The lower the sample volume, the higher the percentage of the total volume is overage. To minimize sample loss, Agilent recommends diluting small volume samples. Note: Setting the volume to zero skips all Load Samples tasks except syringe washing. Flow rate (L/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding. Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges. Default: 5 Practical:

210 (5 L cartridges) 520 (25 L cartridges)

Range: 0.1500 Wash cycles. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the sample flow-through from the Load Samples step is dispensed in the Flow Through Collection plate. If this step is not selected, the flow-through from the Load Samples step dispensed in the Organic Waste plate. The Collect Flow Through step is skipped if the Load Samples step is not conducted. This step is selected by default.

Protocol step Guidelines and notes

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Cup Wash This step removes the residual sample liquid that may remain above the resin bed after sample loading. The Cup Wash step aspirates Cartridge Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the Organic Waste plate, and then washing the syringes at the wash station. This step is selected by default. Volume (L). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 L may offer little benefit. Default: 25 Practical: 2550 Range: 0100 Note: Setting the volume to zero skips all Cup Wash tasks. Wash cycle. Each cycle comprises one cup wash and one syringe wash. Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Internal Cartridge Wash

This step uses Cartridge Wash Buffer to wash non-specifically bound molecules from the resin bed. In preparation for the Internal Cartridge Wash, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Cartridge Wash Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. For the wash operation, this step aspirates the Cartridge Wash Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Flow Through Collection plate or Organic Waste plate. The exterior of the cartridge tips are washed at the wash station to remove any remaining buffer on the cartridge exterior, the cartridges are parked at the seating station, and the syringes are washed at the wash station. If the Load Samples step is selected, the first 5 L (5 L cartridges) or 25 L (25 L cartridges) of Cartridge Wash Buffer is dispensed as a sample chase at the Load Samples flow rate. Next, the Internal Cartridge Wash volume minus the chase volume is dispensed at the Internal Cartridge Wash flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. This step is selected by default. Volume (L). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing. Volume for 5 L cartridges:

Default: 50 Practical: 50100 Range: 0250

Volume for 25 L cartridges: Default: 250 Practical: 250 Range: 0250

Note: Setting the volume to zero skips all Internal Cartridge Wash tasks except for syringe washing. Flow rate (L/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 L/min may not equilibrate through the pores in the beads, resulting in incomplete washing. Default: 10 Practical: 520 Range: 0.5500

Protocol step Guidelines and notes

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Internal Cartridge Wash (continued)

Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 3 Practical: 25 Range: 010

Collect Flow Through

If this step is selected, the flow-through from Internal Cartridge Wash step is dispensed into the Flow Through Collection plate. If the Collect Flow Through step is not selected, the flow-through from the Internal Cartridge Wash step is dispensed into the Organic Waste plate. This step is not selected by default.

Stringent Syringe Wash

This step cleans the syringes with the Syringe Wash Buffer prior to elution. The Stringent Syringe Wash step aspirates the Syringe Wash Buffer into the syringes, draws the buffer through a full syringe stroke to ensure the entire syringe is rinsed, and then dispenses the buffer into the Organic Waste plate. The syringes are then washed at the wash station. This step is selected by default. Volume (L). Volumes higher than the default volume are unlikely to improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing. Default: 50 Practical: 50100 Range: 0250 Note: Setting the volume to zero skips all Stringent Syringe Wash tasks. Wash cycle. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station. Default: 2 Practical: 25 Range: 010

Protocol step Guidelines and notes

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Elute This step uses Elution Buffer to elute bound proteins from the cartridges. In preparation for elution, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate, 10 L of Elution Buffer is aspirated into the syringes and then dispensed into the cartridge cups to prevent potential air gaps from being introduced when the cartridges are seated on the syringe probes. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station. After the elution, the samples are mixed in the Eluate Collection plate and the syringes are washed at the wash station. Note: If the total volume in the Eluate Collection plate is <15 L, the samples will not be mixed. You can also select the Eluate Discard and Add to Flow Through substeps, which are described in the following rows of this table. This step is selected by default. Volume (L): The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. If a strong Elution Buffer is used, the minimum volume is approximately 23 column volumes (1015 L for 5 L cartridges, or 5075 L for 25 L cartridges). The default volumes are conservative and significantly higher than the minimum expected with a strong Elution Buffer. Note: The Eluate Collection plate must be able to accommodate the total volume, which is determined by summing the net elution volume (Elute volume - Eluate Discard volume) and the Existing Collection Volume. For labware-specific maximum well volumes, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Volume for 5 L cartridges:

Default: 25 Practical: 1030 Range: 0250

Volume for 25 L cartridges: Default: 125 Practical: 50150 Range: 0250

Note: Setting the volume to zero skips all Elute tasks except syringe washing.

Protocol step Guidelines and notes

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Flow rate (L/min). A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 L/ min for a given volume of elution buffer (that is, more elution buffer may be required to get the same elution yield at high elution flow rates relative to using lower flow rates for a given elution volume). Default: 5 Practical: 515 Range: 0.1500 Wash cycle. The number of syringe wash cycles to perform at the end of this step. 250 L of DI water is used for each syringe wash cycle. Default: 1 Practical: 13 Range: 010

Eluate Discard This substep of the Elute step permits a specified volume of the eluate from the cartridges to be discarded before the eluate starts to be collected during the Elute step. The Elute step aspirates the Elution Buffer into the syringes, mounts the cartridges, and then dispenses the Elution Buffer at the Elute flow rate through the cartridges. If the Eluate Discard step is selected, the specified volume is dispensed into the Organic Waste plate or Flow Through Collection plate (if the Add to Flow Through step is selected). The remaining Elution Buffer is dispensed through cartridges at the Elute flow rate into the Eluate Collection plate.

Example: If the Elute, Eluate Discard, and Add to Flow Through steps are all selected with the following settings: Elute volume = 15 L (5 L cartridges) or 40 L (25 L cartridges) Eluate Discard volume = 2 L (5 L cartridges) or 10 L (25 L cartridges) the first 2 L (5 L cartridges) or 10 L (25 L cartridges) eluate from the cartridges will be discarded into the Flow Through Collection plate, and the remaining 13 L (5 L cartridges) or 30 L (25 L cartridges) eluate will be collected in the Eluate Collection plate.

Select the Eluate Discard step in situations where minimizing the volume of eluate is important. For AssayMAP cartridges, the initial elution volume (~2 L for 5 L cartridges or ~10 L for the 25 L cartridges) contains small or no measurable amounts of analyte. This step is not selected by default. Volume (L). The first volume of eluate that will be discarded during the Elute step. This value can equal, but cannot exceed the Elute volume. Default: 0 Practical:

5 L cartridges: 02 25 L cartridges: 010

Range: 0250

Protocol step Guidelines and notes

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Add to Flow Through

If selected, this step dispenses the Eluate Discard volume into the Flow Through Collection plate. If the Add to Flow Through step is not selected, the Eluate Discard is dispensed into the Organic Waste plate. This step is not selected by default. Note: The Add to Flow Through step is an option only if both the Elute and Eluate Discard steps have been selected.

Existing Collection Volume

This step enables users to specify an amount of liquid that is in the wells of the Eluate Collection plate at the beginning of the run. The Existing Collection Volume and the net volume from the Elute step (Elute Volume - Eluate Discard Volume) feeds into logic that adjusts the well-bottom offset for sample elution, calculates the eluate mixing volume, and dynamically moves the head in and out of the wells during elution and eluate mixing in a volume-dependent manner. For the maximum practical working volumes of labware for eluate collection, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Select this step when the Eluate Collection plate contains a volume of liquid useful for immediately diluting the eluates, for adjusting the pH of the eluates, or to aid in the recovery of small volumes of eluates from AssayMAP cartridges. Volume (L): Default: 0 Practical: 0250 Range: 0300

Final Syringe Wash This step uses the wash station to flush potential contaminants from the syringes. Before the final syringe wash begins, 20 L of air is aspirated into the syringes, the probes go into the cartridge cups to a depth that is just short of the normal engagement position, liquid in the cups is removed by a 60 L aspiration and then discarded into the Organic Waste plate. No solution is added into the cartridge cups. Note: If the Final Syringe Wash is skipped, the 10 L of elution buffer will remain in the cartridge cups. During each Final Syringe Wash cycle, the head aspirates 250 L into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste. Note: In cases where carryover is a major concern, increasing the number of wash cycles may provide improved washout, but with a cost of increased assay time and reduced syringe lifetime. The best practice is to use the Syringe Wash utility to wash the syringes between runs with stringent wash solutions. This step is selected by default. Wash Cycles: Default: 3 Practical: 35 Range: 010

Protocol step Guidelines and notes

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Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Protein Cleanup protocol using the default method. Changing the selections or parameters will alter the movements.

Protocol step Head moves to deck location...

Action

Starting protocol 2 Parks all cartridges that might have been loaded on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

Initial Syringe Wash 1 Washes the syringes.

Prime 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

5 Aspirates 10 L of Priming Buffer for the cartridge air-gap prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

5 Aspirates the Priming Buffer.

2 Mounts the cartridges onto the head.

3 Dispenses the buffer through the cartridges into the Organic Waste plate to prime the cartridges.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

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Equilibrate 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

8 Aspirates 10 L of Equilibration Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Equilibration Buffer.

2 Mounts the cartridges on the head.

3 Dispenses the Equilibration Buffer through the cartridges to equilibrate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Load Samples 4 Aspirates the samples into the syringes.

1 Washes the exterior of the syringe probes.

2 Mounts the cartridges on the head.

7 Dispenses the samples through the cartridges and into the Flow Through Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Cup Wash 8 Aspirates the Cartridge Wash Buffer.

2 Washes the cartridge cups and exercises the cartridges off task.

3 Dispenses the Cartridge Wash Buffer into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Internal Cartridge Wash

2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

8 Aspirates 10 L of Cartridge Wash Buffer for the cartridge air- gap prevention step.

2 Dispenses the 10 L of buffer into the cartridge cups and exercises the cartridges off task.

8 Aspirates the Cartridge Wash Buffer into the syringes for the sample chase and the Internal Cartridge Wash steps.

2 Mounts the cartridges on the head.

3 Dispenses 5 L (5 L cartridges) or 25 L (25 L cartridges) Cartridge Wash Buffer through the cartridges at the Load Samples flow rate for the sample chase step.

3 Dispenses the remaining Cartridge Wash buffer through the cartridges at the Internal Cartridge Wash flow rate and into the Organic Waste plate.

2 Parks the cartridges in the seating station.

1 Washes the syringes.

Stringent Syringe Wash

5 Aspirates the Priming & Syringe Wash Buffer.

3 Dispenses the buffer.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Elute 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the exterior of the syringe probes.

6 Aspirates 10 L of Elution Buffer for the cartridge air-gap- prevention step.

2 Dispenses the 10 L of Elution Buffer into the cartridge cups and exercises the cartridges off task.

6 Aspirates the Elution Buffer.

2 Mounts the cartridges on the head.

9 Elutes the samples into the Elution Collection plate.

1 Washes the exterior of the cartridge tips.

2 Parks the cartridges in the seating station.

9 Mixes the eluates.

1 Washes the syringes.

Final Syringe Wash 2 Aspirates 20 L of air above this location, moves down to just above the cartridge engagement point and aspirates 60 L, and then exercises the cartridges off task.

3 Dispenses into the Organic Waste plate.

1 Washes the syringes.

Protocol step Head moves to deck location...

Action

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Reference library

1 Bovee, M., Krahenbuhl, A. & Murphy, S., Rapid Antibody Digestion Enabled by Automated Reversed-Phase Desalting on the Agilent AssayMAP Bravo Platform, Agilent Application Note 5991-6478EN, 2016

See the Agilent AssayMAP Bravo Citation Index for published papers that used the AssayMAP Bravo Platform.

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15 Utilities User Guides Normalization, Reformatting and Serial Dilution utilities

Other utilities

Normalization v3.0 User Guide on page 585

Reformatting v3.0 User Guide on page 623

Serial Dilution v3.0 User Guide on page 653

Cartridge Transfer v2.0 User Guide on page 506

Pipette Tip Transfer v2.0 User Guide on page 512

Reagent Aliquot v2.0 User Guide on page 518

Reagent Transfer v3.0 User Guide on page 525

Single Liquid Addition v2.0 User Guide on page 542

Syringe Test v2.0 User Guide on page 549

Syringe Wash v3.0 User Guide on page 567

System Startup/Shutdown v3.0 User Guide on page 574

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Cartridge Transfer v2.0 User Guide

The Cartridge Transfer utility moves full columns of AssayMAP Cartridges from the source cartridge rack, which is a standard 96AM Cartridge Rack and Receiver Plate at deck location 6, to the destination cartridge rack, which is the 96AM Cartridge and Tip Seating Station at deck location 2.

Before you start

The following figure shows the required deck layout.

On the AssayMAP Bravo deck, make sure: The empty seating station is at deck location 2. The 96AM Cartridge Rack and Receiver Plate is at deck location 6. It may be

completely or partially filled with cartridges. Make sure you remove the lid. The cartridges are arranged in full columns of eight cartridges each (if the source

96AM Cartridge Rack and Receiver Plate is partially filled). The 96AM Wash Station or the later model 96 Channel Wash Station is at deck

location 1. All other deck locations are empty.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded.

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To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Cartridge Transfer utility.

To set up the protocol: 1 Open the Utility Library. 2 Locate Cartridge Transfer, and then click Utility.

The Cartridge Transfer utility opens.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the Cartridge Transfer run on page 509.

To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the following settings for the run:

7 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Cartridge Transfer run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories and cartridge rack are at the assigned deck locations,

as shown in the Deck Layout image of the form.

Setting Description First Column in Cartridge Rack The first available column, counting from

the left side of the 96AM Cartridge Rack and Receiver Plate. The value must be between 1 and 12.

Number of Columns to Transfer The number of columns of cartridges that you want to transfer to the seating station. The value must be between 1 and 12.

First Column in Seating Station The first column, counting from the left side, of the seating station to which cartridges will be transfered.

Reverse Process The option to return the moved cartridges back to their original locations. See Returning the cartridges to their original locations on page 510.

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CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box. For a summary of the movements, see Automation movements during the Cartridge Transfer protocol on page 511.

When the run is finished, remove the 96AM Cartridge Rack and Receiver Plate with any unused cartridges from deck location 6.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Returning the cartridges to their original locations

If you have accidentally transferred cartridges to the wrong columns of the seating station, you can return the cartridges to their original locations in the 96AM Cartridge Rack and Receiver Plate. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

p

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To return the cartridges to their original locations: 1 Without changing any other settings from their original transfer process, select the

Reverse Process check box. VWorks Plus. You must save the method before you can run it.

2 To save the Reverse Process method:

a Click .

b In the Save File As dialog box, type the file name and click Save. 3 In the form, click Select Method.

In the Open File dialog box, select the Reverse Process method that you created in step 2, and click Open.

4 Click to reverse the original cartridge transfer process.

Automation movements during the Cartridge Transfer protocol

This section describes the basic movements of the AssayMAP Bravo Platform during a generic forward Cartridge Transfer protocol. Changing the selections or parameters will alter the movements.

Protocol step

Head moves to deck location...

Action

1 6 Loads the desired number of cartridges.

2 2 Parks the cartridges in the seating station.

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Pipette Tip Transfer v2.0 User Guide

The Pipette Tip Transfer utility moves 112 columns of Agilent 250-L pipette tips from a source tip box at AssayMAP Bravo deck location 6 into an empty 96AM Cartridge & Tip Seating Station at deck location 2. This utility is designed to transfer contiguous, full columns of tips, where each column has eight pipette tips.

Before you start

The following figure shows the required deck layout.

On the AssayMAP Bravo deck, make sure: The empty seating station is at deck location 2. An Agilent 250-L tip box is properly positioned at deck location 6. The tip box may

be full or partially filled with pipette tips. For partially filled tip boxes, ensure that the tips are arranged in contiguous, full columns of eight pipette tips each. Make sure that the tip box lid has been removed.

The 96AM Wash Station or the later model 96 Channel Wash Station is at deck location 1.

All other deck locations are empty.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded.

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To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Pipette Tip Transfer utility.

To set up the protocol: 1 Open the Utility Library. 2 Locate Pipette Tip Transfer, and then click Utility.

The Pipette Tip Transfer utility opens.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the Pipette Tip Transfer run on page 515.

To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the following settings for the run:

7 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Pipette Tip Transfer run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

Setting Description Columns of pipette tips in the Source Tip Box

Specifies the range of pipette tips initially present in the pipette tip box before the run starts.

Left field is the first column that contains tips. Right field: is the last column that contains tips. For example: For a full tip box:

left field = 1, right field = 12 For partially filled tip box with tips in columns 6 - 10:

left field = 6, right field = 10

IMPORTANT The pipette tip box must contain contiguous columns of 8 pipette tips each, within the range of columns specified.

Columns to be filled in the Seating Station

Specifies the range of wells to be filled with tips in the seating station at deck location 2.

In the left field, specify the column number of the first column in the seating station that will contain tips after completing the transfer.

In the right field, specify the column number of the last column in the seating station that will contain tips after completing the transfer.

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To start the protocol run: 1 Ensure that the accessories and pipette tip box are at the assigned deck locations,

as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

CAUTION A collision can occur if any pipette tips are present in the columns outside the specified range.

2 Click to start the run.

To monitor the progress of the run, check the Status box. For a summary of the movements, see Automation movements during the Pipette Tip Transfer protocol on page 517.

When the run is finished, remove the Pipette Tip Box from deck location 6 from the deck.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Automation movements during the Pipette Tip Transfer protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Pipette Tip Transfer protocol using the Default Method settings. Changing the selections or parameters will alter the movements.

Protocol step

Head moves to deck location...

Action

1 1 Dispenses any liquid remaining in the syringes into the wash station.

2 6 Presses on the pipette tips while the head is centered above the tip box, even if the tip box is only partially full. Note: A collision will result if the head is offset from the tip box location when pressing on pipette tips.

3 2 Ejects the pipette tips into the seating station.

4 2 If applicable, picks up any columns of excess pipette tips from the seating station. The number of excess tips is calculated from the Application Settings. If Application Settings specify that the number of columns of pipette tips in the tip box matches the number of columns of pipette tips to be transfered into the seating station, this step is skipped. Note: The head is offset to the right of the seating station, instead of centered above the station, when picking up excess columns of pipette tips.

5 6 If applicable, ejects any excess pipette tips back into the tip box. Note: Excess tips are always returned to the left side of the tip box, starting at column 1.

6 2 If applicable, picks up pipette tips from the seating station and moves them to the columns specified in the form. If the pipette tips are already in the correct location after the initial transfer, this step is skipped.

7 1 Moves to a safe height above the wash station.

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Reagent Aliquot v2.0 User Guide

The Reagent Aliquot utility aliquots a reagent from a single column or reservoir of a Bulk Reagent storage plate into 1 to 12 columns of a 96-well microplate (Reagent Aliquot plate). The utility uses a single column of eight Agilent 250-L pipette tips to prepare the aliquots. This utility is a useful starting point for most applications and workflows, because it simplifies the process of preparing reagent plates for an automation run.

Before you start

Labware The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view)

Labware options Manufacturer part number* Deck location

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4, 7

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 4, 7

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4, 7

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Reagents When preparing a reagent plate for another application run, make sure to use the

application-specific Reagent Volume Calculator to: Determine the amount of reagent or sample required in each well. Make appropriate labware selections. Calculate bulk reagent volumes required, including required overage volumes.

Prepare appropriate volumes of bulk reagents, and manually prepare a Bulk Reagent Plate.

Prepare the Bulk Reagent Plate with the intended reagents. Pipette an excess volume in the plate of at least: PCR plates: 1520 L per well U-bottom, V-bottom, and flat-bottom microplates: 2530 L per well Deep-well plates: 3545 L per well Reservoir plates: varies depending on type

For details, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Note: These overage volumes are higher than in other applications because this utility uses reverse pipetting.

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 4, 7

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 4, 7

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 4, 7

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 4, 7

96 Costar 3363, PP Conical Bottom Corning Costar 3363 4, 7

96 Nunc 269620, Flat Bottom, Polystyrene Thermo-Fisher 269620 4, 7

96 Eppendorf 96-500 V-bottom, Clear, PolyPro Eppendorf 96/500 4, 7

96 Eppendorf 96-1000 U-bottom, Clear, PolyPro Eppendorf 96/1000 4, 7

96 Waters 186005837, Clear PolyPro Waters 186005837 4, 7

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 7

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 7

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom

Agilent 201254-100 7

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96-LP

7

96 V11 Manual Fill Reservoir Agilent G5498B#049 7

*For dimensionally equivalent alternatives, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Labware options Manufacturer part number* Deck location

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CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Reagent Aliquot utility.

To set up the protocol: 1 Open the Utility Library. 2 Locate Reagent Aliquot, and then click Utility.

The Reagent Aliquot utility opens.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

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To run the selected method, go to Starting the Reagent Aliquot run on page 523.

To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the following settings for the run:

7 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Setting Description Columns of Pipette Tips in the Pipette Tip Box

Specifies the range of pipette tips present in the pipette tip box before the run starts.

In the left field, specify the column number of the first column of pipette tips present in the tip box.

In the right field, specify the column number of the last column of pipette tips present in the tip box.

One full column of pipette tips is used for every run of this utility. The remaining pipette tips are automatically returned to the pipette tip box for use in future runs.

IMPORTANT The pipette tip box must contain contiguous columns of 8 pipette tips each, within the range of columns specified.

Columns To Receive Aliquots in the Reagent Aliquot Plate

Specifies the range of wells that you want to fill in the Reagent Aliquot Plate.

In the left field, specify the column number of the first column in the Reagent Aliquot Plate that will be filled with aliquots.

In the right field, specify the column number of the last column in the Reagent Aliquot Plate that will be filled with aliquots.

Aliquot Reagent Source Column from the Bulk Reagent Plate

Defines the column of the Bulk Reagent Plate that contains the bulk reagent to be aliquoted into the Reagent Aliquot Plate.

Aliquot Volume (L)

Defines the volume of bulk reagent to be aliquoted to each of the selected wells in the Reagent Aliquot Plate. Note: The overage volume is not automatically included in this value. If overage is required for your application, ensure that this value is adjusted to include overage.

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Starting the Reagent Aliquot run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories, filled reagent plates, and collection plates are at the

assigned deck locations, as shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

CAUTION A collision can occur if any pipette tips are present in the columns outside the specified range.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

When the run is finished, remove the labware from the deck.

WARNING To stop a run in an emergency, use the emergency stop pendant.

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To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Reagent Aliquot protocol using the Default Method settings. Changing the selections or parameters will alter the movements.

Protocol step

Head moves to deck location...

Action

1 1 Dispenses any liquid remaining in the syringes into the wash station.

2 2 Performs Tips Off to ensure no tips remain on the head at the beginning of the run.

3 1 Performs two syringe drying cycles by aspirating and dispensing air and then conducting at tip touch across the wash station chimneys.

4 6 Presses on all the pipette tips in the tip box. Although all the pipette tips are picked up from the tip box at this step, only 1 column of pipette tips is used for this utility.

5 2 Ejects the pipette tips into the seating station. Note: The head is centered over the seating station when ejecting the pipette tips.

6 2 If applicable, picks up any columns of excess pipette tips from the seating station. Note: The head is offset to the right side of the seating station when picking up columns of excess pipette tips.

7 6 If applicable, ejects the excess pipette tips back into the tip box. Note: Excess pipette tips are always returned to the left side of the tip box, starting at column 1.

8 2 Presses on a single column of pipette tips from the seating station using the left- most column of syringes.

9 7 Aspirates bulk reagent into the pipette tips from the Bulk Reagent Plate. Note: This step uses reverse pipetting, which involves aspirating a small excess of liquid into the pipette tips to ensure accurate pipetting for the final aliquot of a set.

10 4 Dispenses the reagent into the Reagent Aliquot Plate. The utility uses either of the following two dispense modes: Multiple low-volume aliquots are dispensed from a single aspirate step. A single high-volume aliquot requires multiple aspirate steps. In both cases, a small excess volume of the bulk reagent remains in the pipette tips because of the reverse pipetting technique used.

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Reagent Transfer v3.0 User Guide

The Reagent Transfer v3.0 utility transfers samples or reagents from a Source Plate to a Destination Plate. This utility is useful for a wide variety of sample manipulation and reagent preparation operations. The utility features include the ability to: Conduct liquid transfers using either the bare probes of the AssayMAP head or

250-L pipette tips. Control the liquid-handling processes, including:

Use reverse pipetting or forward pipetting modes, which can increase pipetting accuracy depending on the liquid properties and volume being transferred.

Select from a list of liquid classes or enter a custom liquid class. Specify whether to conduct syringe drying cycles and pre-wet cycles.

Control the locations of the liquid transfer in both the Source and Destination plates.

Before you start

Labware The following table provides a complete list of labware options and the corresponding deck locations for the Source Plate and Destination Plate. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

11 7 & 4 Step 9 and Step 10 are repeated until all aliquots have been prepared in the Reagent Aliquot Plate.

12 7 Dispenses excess reagent within the pipette tips back into the Bulk Reagent Plate.

13 2 Ejects the used pipette tips back into the seating station. Note: The used pipette tips are always ejected to column 1 of the seating station.

14 1 Moves the head to a safe distance above the wash station.

Protocol step

Head moves to deck location...

Action

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Figure Labware locations on the Bravo deck (top view)

Labware options for deck locations 7 and 8 Manufacturer part number* Source Plate volume overage (well)**

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 10 L

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 10 L

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 10 L

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 30 L

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 20 L

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 20 L

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 20 L

96 Costar 3363, PP Conical Bottom Corning Costar 3363 20 L

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 25 L

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 3 mL

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 4.5 mL

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom

Agilent 201254-100 20 mL

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96-LP

20 mL

96 V11 Manual Fill Reservoir Agilent G5498B#049 35 mL

96AM Receiver Plate Included with Agilent cartridge rack

Not applicable

96 Eppendorf 96-500 V-bottom, Clear, PolyPro*** Eppendorf 96/500 25 L

96 Eppendorf 96-1000 U-bottom, Clear, PolyPro*** Eppendorf 96/1000 25 L

96 Waters 186005837, Clear PolyPro*** Waters 186005837 30 L

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Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Reagent Transfer utility.

To set up the protocol: 1 Open the Utility Library. 2 Locate Reagent Transfer, and then click Utility.

*For dimensionally equivalent alternatives, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. **This overage does not include the volume required for reverse pipetting. The volume is listed by well, where a well is defined as an individual well in a 96-well plate. But in labware where multiple syringes can draw from a common source, a well is defined as that common source. For example, a 12-Column Low-Profile Reservoir has 12 wells, an 8-Row Low-Profile PolyPro Reservoir has 8 wells, and a Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir has only 1 well. ***Pipette tips are required for reagent transfers using these deep-well plate options.

Labware options for deck locations 7 and 8 Manufacturer part number* Source Plate volume overage (well)**

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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The Reagent Transfer utility opens.

3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Reagent Transfer run on page 536.

To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the following settings for the run:

a Specify the Pipette Tip Settings.

Pipette Tip Setting Description Use Pipette Tips The option to use 250 L pipette tips to transfer the

liquid: To use pipette tips, select the check box. To use the bare probes on the AssayMAP head,

clear the check box. Note: Ensure that the labware selection is appropriate for the bare probes. For details, see Before you start.

Default: Not selected

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b Specify the Source Plate Settings.

Transfer Pipette Tips from Deck Location 6

If the Use Pipette Tips check box is selected, specify whether to transfer pipette tips using the Reagent Transfer utility. CAUTION A collision can occur if any pipette tips are present in the source tip box outside of the range specified on the form. To transfer the pipette tips from a source tip box at

deck location 6 to the empty seating station at deck location 2, select the check box.

IMPORTANT The pipette tip box must contain contiguous columns of 8 pipette tips.

To use pipette tips that are already present in the seating station at deck location 2, clear the check box. For example, if you do a run and transfer the pipette tips from deck location 2 to the seating station, and then want to do additional runs with the same settings, the pipette tips can be reused.

IMPORTANT The pipette tips must be in contiguous columns of 8 pipette tips.

Default: Not selected Columns of Pipette Tips at Deck Location 6

If the Transfer Pipette Tips from Deck Location 6 check box is selected, specify the range of full columns of pipette tips present in the source tip box (deck location 6) at the beginning of the run: Left field is the first column that contains tips. Right field is the last column that contains tips. For example: For a full tip box:

left field = 1, right field = 12 For partially filled tip box with tips in columns 6 - 10:

left field = 6, right field = 10 Default: 1 to 12 Range: Starting column: 112 Ending column: 112

Pipette Tip Setting Description

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Source Plate Setting Description Columns of Samples to Transfer

The range of wells (full columns) in the Source Plate (deck location 7) to be used for the reagent transfer. Note: If using bare probes, use the default range of 1 to 12. When using bare probes, the head moves as if it is moving all 96 positions from the Source Plate to the Destination Plate. Therefore, liquid will be moved from every position on the Source Plate where it exists to every corresponding position on the Destination Plate. Default: 1 to 12 Range: Starting column: 112 Ending column: 112

Initial Well Volume The volume of liquid present in the wells of the Source Plate (deck location 7) before transferring the liquid. For labware where multiple syringes draw from a common source, a well is defined as that common source. For example, a 12-Column Low-Profile Reservoir has 12 wells, an 8-Row Low-Profile PolyPro Reservoir has 8 wells, and a Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir has only 1 well. For these labware types, the Initial Well Volume is calculated as:

For example, in the Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir the overage required is 20 mL plus 96 times the volume to be transferred (e.g., 100 L) divided by 96:

This value is used to calculate the appropriate height for the pipette tips or probes during the aspiration process, and to facilitate proper dynamic-tip behavior. Note: Using a value of 0 will disable dynamic-tip movement and force the probes or tips to aspirate from the minimum distance from the well bottom of the selected labware. This might result in the pipette tip or probe exterior being exposed to liquid that might not be removed at the wash station. Default: 0 (L) Range: 01500 (L)

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c Specify the Destination Plate Settings.

Volume to Transfer The volume of liquid per syringe to transfer from the Source Plate (deck location 7) to the Destination Plate (deck location 8). If this volume setting is greater than the capacity of the probes or pipette tips, the transfer will be evenly split into multiple aspirate-and-dispense cycles. Default: 10 (L) Range: 01200 (L) Note: The Automatic Liquid Class setting is designed for a 5 L minimum. A volume less than 5 L might require a custom liquid class. Note: A value of 0 will drive the probes or tips to the minimum height within the wells and keep them there for the duration of the aspiration step.

Source Plate Setting Description

Destination Plate Setting

Description

Columns to Receive Samples

The range of full columns in the Destination Plate (deck location 8) to which the samples will be transferred. When using pipette tips, this range can differ from the range specified for the Columns of Samples to Transfer value, but it must contain the same number of full columns. If not, the software displays an error message, and the protocol automatically aborts. When using bare probes, use the default range of 1 to 12. The AssayMAP Bravo head moves as if it is transferring liquid from all 96 wells. The wells that contain liquid dictate which wells are transferred. Default: 1 to 12 Range: Starting column: 112 Ending column: 112

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d Specify the Wash Settings.

Initial Well Volume The volume of liquid present in each well of the Destination Plate (deck location 7) before starting the transfer. Note: A well is defined as an individual well in a 96-well plate. But in labware where multiple probes can pull from the same continuous liquid source, a well is defined as that continuous liquid source. For example, a 12-Column Low-Profile Reservoir has 12 wells, an 8-Row Low-Profile PolyPro Reservoir has 8 wells, and a Seahorse 201254-100, PP, No Walls, Pyramid-bottom Reservoir has only 1 well. This value is used to calculate the appropriate height for the pipette tips or probes during the transfer dispense process, and to facilitate proper dynamic tip behavior. Default: 0 (L) Range: 01500 (L)

Mix Cycles after Transfer

The number of aspirate-and-dispense cycles used to mix the contents of the Destination Plate (deck location 8) after the transfer has been completed. The volume used for the mixing process is automatically calculated based on practical low-volume pipetting limits and maximum syringe or probe volume capacities. Default: 0 Range: 020 Mixing formula:

If (x 50 L) > Syringe/Tip Capacity, then Mixing Volume = Syringe/Tip Capacity If 50 L x Syringe/Tip Capacity, then Mixing Volume = 0.75x If x < 50 L, then Mixing Volume = 0.5x where, x is the final volume in the destination wells, maximum Syringe Capacity = 250L, maximum Tip Capacity = 140L Note: Syringe and pipette tip capacities vary depending on the Blowout Volume setting.

Destination Plate Setting

Description

Wash Setting Description Initial Syringe/Tip Wash Cycles

The number of syringe or pipette tip wash cycles to be performed at the wash station (deck location 1) before starting the reagent transfer portions of the run. Default: 0 Range: 010

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e Specify the Liquid Handling Preferences.

Final Syringe/Tip Wash Cycles

The number of syringe or pipette tip wash cycles to be performed at the wash station (deck location 1) after completing the reagent transfer portions of the run. Default: 0 Range: 010

Wash Setting Description

Liquid Handling Setting

Description

Syringe Drying Cycles The number of syringe purge cycles to be conducted with air before starting the run. This step occurs at different times for runs that use pipette tips instead of probes. If using pipette tips. This step occurs before the Initial

Wash cycles. If using bare probes. This step occurs after the Initial

Wash Cycles are completed. Default: 2 Range: 05

Pre-Wet Cycles The number of times to wet the probes or pipette tips with the liquid from the Source Plate (deck location 7) before starting the transfer process. Note: Prewetting the pipette tips is a common pipetting technique that can greatly increase accuracy in certain situations. Default: 0 Range: 05

Reverse-Pipetting Overage

Specifies whether to run in reverse pipetting mode, and if so, the volume to be added to the volume that is being transferred. To specify forward pipetting mode. Use the default

setting of 0 L. To specify reverse pipetting mode. Use a value that is

greater than 0 L, and select the discard location for the extra liquid. This extra liquid remains in the pipette tips or probes for the entire reagent transfer process. Before conducting the mix cycles after the transfer, the excess volume is discarded to the Source Plate (deck location 7) or to Wash Station Waste (deck location 1), depending your selection. Note: Reverse pipetting is a common pipetting technique that can greatly increase accuracy in certain situations.

Default: 0 (L) Range: 050 (L)

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f In the Labware Table, select that labware you are using at deck locations 7 and 8.

7 To save the method:

Blowout Volume The volume of air to dispense out of the syringes or pipette tips after completing the liquid transfer steps. The blowout process is different for the reverse-pipetting and forward-pipetting modes because of the presence of the Reverse-Pipetting Overage volume. Forward pipetting mode. The blowout occurs after

the transfer has completed, and then again after mixing, if conducted.

Reverse pipetting mode. The blowout occurs only after the mixing step, if conducted, because of the presence of the Reverse-Pipetting Overage volume.

Default: 0 (L) Range: 050 (L)

Liquid Class The pipetting speed and accuracy. To ensure consistent pipetting, always select a liquid class for liquid-handling tasks. Choose from the following options: (default). Automatically assigns one of

the following liquid classes, based on whether pipette tips are used and the volume being transferred. These are good general-purpose liquid classes for most reagents and buffers that are used for the AssayMAP system. Bare probes:

020 L (AM_ProbesLowVol) > 20 L (AM_ProbesHighVol)

Pipette tips: 020 L (AM_250uLTipsLowVol) > 20 L (AM_250uLTipsHighVol)

Slow Flow (5 L/sec). A slower flow rate is better for viscous solutions.

Fast Flow (100 L/sec). A faster flow rate is a good starting point for high organic solutions.

If these flow rates do not provide the desired performance, you may enter a custom liquid class. To create a custom liquid class, you use the VWorks Liquid Library Editor. For details on how to open the VWorks Liquid Library Editor, see Creating a custom liquid class.

To enter a custom liquid class for the reagent transfer: In the Liquid Class box, type the liquid class name exactly as it appears in the VWorks Liquid Library Editor.

Liquid Handling Setting

Description

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a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Reagent Transfer run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

2 If the method specifies Use Pipette Tips, do one of the following: If the Transfer Pipette Tips from Deck Location 6 check box is selected, ensure

that the: Pipette tip box containing the specified range of pipette tips is in

position at deck location 6. Empty seating station is in place at deck location 2.

If the Transfer Pipette Tips from Deck Location 6 check box is cleared, ensure that the seating station at deck location 2 contains the specified range of pipette tips. Note: A message will appear and ask you to verify that the pipette tips are in the correct locations. Ensure that the pipette tips are arranged in the specified locations.

CAUTION A collision can occur if any pipette tips are present in the columns outside the specified range.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

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3 Click to start the run.

To monitor the progress of the run, check the Status box.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. See the following section for guidance on how to handle error, warning, and verification messages. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Error, warning, and verification messages

Detected error in the setup If the software detects a conflict in the setup, an error message or warning will appear before the run begins.

Figure Examples of error messages that prevent run execution

To resolve error messages about setup conflicts: 1 In the form, resolve the settings described in the error message, for example:

Insufficient number of columns of pipette tips

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Unequal number of columns selected for Source and Destination plates Incompatible labware selections (labware that require pipette tips) VWorks Plus. Administrator or technician privileges are required to create and modify methods.

2 To save the new method, click .

In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

3 To select the new method, click Select Method. In the Open File dialog box, select the new method, and click Open.

4 Click to start the run.

Verification and warning messages Verification and warning messages that appear allow you to continue with the run as specified, while assuming the risk posed by the warning. The following figure shows some examples.

Figure Examples of verification messages

To resolve verification and warning messages: Follow the on-screen instructions to verify that the settings are correct, and then do one of the following: If the settings are correct, click Continue to start the run.

Note: A Pipette Tip Verification message appears whenever pipette tips are used.

If you need to change a setting: VWorks Plus. Administrator or technician privileges are required to create and modify methods. a Click Pause and Diagnose. The Scheduler Paused dialog box opens. b In the Scheduler Paused dialog box, click Abort process.

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c In the form, change the settings to resolve the conflict. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

d To save the new method, click .

In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

e To select the new method, click Select Method. In the Open File dialog box, select the new method, and click Open.

f Click to start the run.

Creating a custom liquid class

Administrator or technician privileges are required to create or modify liquid classes.

To create a custom liquid class: 1 In the Reagent Transfer v3.0 form, click Toggle Full Screen to change the display of

the form so that the VWorks menubar and toolbar are visible, as the following figure shows.

2 In the VWorks window, click Tools > Liquid Library Editor. The Liquid Library Editor opens. If the menubar is not visible in the VWorks window, right-click the window and select Menubar in the shortcut menu that appears.

3 For instructions on how to use the Liquid Library Editor, see the section on specifying pipette speed and accuracy in the VWorks Version 4 Automation Control Setup Guide in the VWorks Knowledge Base.

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Figure VWorks Tools menu and form displayed with full screen off

Figure VWorks Liquid Library Editor window

Automation movements during the protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Reagent Transfer protocol using the default method settings. Changing the selections or parameters will alter the movements.

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Protocol step

Head moves to deck location...

Action

1 1 Washes the syringes. If pipette tips are used for the transfer, this step occurs after step 3 below to facilitate pipette tip washing.

2 1 Purges the syringe contents to waste for the number of cycles set in the Syringe Drying Cycles setting.

3 6 If applicable, picks up the pipette tips from the source tip box. This step occurs only if the Use Pipette Tips check box and the Transfer Pipette Tips from Deck Location 6 check box are selected.

2 If applicable, ejects the pipette tips into the seating station.

4 2 If applicable, picks up all the pipette tips from the seating station This step occurs only if the Use Pipette Tips check box is selected and the Transfer Pipette Tips from Deck Location 6 check box is cleared. A confirmation message verifies that the pipette tip locations are correct.

5 7 Aspirates and dispenses the liquid in the Source Plate to wet the pipette tips or syringe probes for the number of cycles set in the Pre-Wet Cycles setting.

6 7 Aspirates the volume to be transferred from the Source Plate using the pipette tips or syringe probes.

7 8 Dispenses the liquid into the Destination Plate.

8 7 & 8 Repeats steps 6 and 7 until the full volume specified in the Volume to Transfer setting has been transferred to the Destination Plate. Pipette tip maximum volume: ~140 L can be pulled into a mounted tip before the excess fluid overflows into the syringe, because the probe protrudes into the tip volume capacity. Probe maximum volume: 250 L can be aspirated into the syringe before the syringe is full. The probe maximum volume will be reduced automatically by an amount that is equal to the specified Blowout Volume.

9 8 Mixes the contents of the Destination Plate.

10 8 Performs a blowout slightly above the liquid level of the Destination Plate to clear any remaining liquid.

11 8 Performs a tip touch on the left and right sides of the wells in the Destination Plate.

12 1 Washes the pipette tips or syringe probes.

13 2 If pipette tips are used, ejects the pipette tips into the seating station

14 1 Moves the head above the wash station.

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Single Liquid Addition v2.0 User Guide

The Single Liquid Addition Utility is specifically designed to adjust the pH of samples after the samples have been digested using the In-Solution Digestion: Multi-Plate application. This utility enables you to rapidly adjust buffer composition or add a component to up to four sample plates by transferring liquid from a common reagent plate, to up to four independent sample plates. An optional stringent syringe wash step is included to prevent carry over.

Before you start

Labware The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Labware locations on the Bravo deck (top view) .

Labware Manufacturer part number*

Deck location options

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 4

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 4

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 4

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 4

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Reagents Make sure you: Prepare a reagent plate that contains the solution that you want to add to the

Sample plates. Place the reagent plate at deck location 4. Prepare the Syringe Wash Buffer. For suggested solutions to use, see Preparing the

solutions in the In-Solution Digestion: Multi-Plate v2.0 User Guide on page 235. Ensure that you have four Greiner 650207 White U-Bottom plates for the wash

plates. You must fill the plates with the Syringe Wash Buffer, and then run the Plate Stacking protocol to stack the wash plates at Bravo deck location 2. For details, see Running the Plate Stacking protocol on page 544.

CAUTION A small reagent volume excess is required in all labware types to ensure proper volume transfer. Use the Reagent Volume Calculator to automatically include excess volume, or look up the recommended values for each labware type in the Labware Reference Guide.

Note: You can find the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 4

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 4

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 2, 4, 5, 6, 8, 9

96 Costar 3363, PP Conical Bottom Corning Costar 3363 4

96 V11 Manual Fill Reservoir Agilent G5498B#049 4

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Labware Manufacturer part number*

Deck location options

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Opening the Single Liquid Addition utility

To open the utility: 1 Open the Utility Library. 2 Locate Single Liquid Addition, and then click Utility.

The Single Liquid Addition utility opens.

Running the Plate Stacking protocol

The Plate Stacking protocol stacks the wash plates properly at Bravo deck location 2 to prevent collision during the Single Liquid Addition protocol run.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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IMPORTANT The Single Liquid Addition protocol requires the stack of four wash plates, even if you have less than four sample plates. Ensure that the wash plates are Greiner 650207 White U-Bottom.

Before running the Plate Stacking protocol, prepare four Greiner 650207 White U-Bottom plates as follows: Fill the plates with 300 L Syringe Wash Buffer. The plates will serve as wash plates

that are used for cleaning the syringes between liquid transfers. Make sure the filled wells correspond to the location of the samples in the sample

plates.

To stack the prepared wash plates:

1 In the Single Liquid Addition form, click . The protocol starts.

2 Follow the on-screen instructions for placing the wash plates at the appropriate locations on the AssayMAP Bravo deck.

3 Check the Status box in the upper right corner of the form. When the Protocol Complete message is displayed, click OK.

Setting up the Single Liquid Addition protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Single Liquid Addition utility.

To set up the Liquid Addition protocol: 1 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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2 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

3 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Single Liquid Addition run on page 547.

To create or modify a method, proceed to step 4. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

4 In the Application Settings area, specify the following properties for the run:

Property Description Number of Initial Syringe Washes

The number of cycles for rinsing syringes with deionized water at the beginning of the run. Range: 010 Default: 3

Number of Sample Plates The number of sample plates (1-4) to which a liquid will be added in this utility. Place only the number of sample plates used on the deck. Positions 2-4 are optional and need not be filled. Range: 14 Default: 1

Initial Volume in Sample Plate

The initial volume of the samples. All plates must have the same sample volume. Range: 0250 Default: 10

Volume to Add to Sample Plate

The volume of solution being added to the samples. This may be limited by the labware. Range: 0250 Default: 50

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5 In the Labware Table, select that labware you are using at deck location 4. 6 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Single Liquid Addition run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck.

Number of Mixing Cycles The number of mixing cycles that will be used to homogenize the sample liquid and the liquid that is added. Range: 020 Default: 10

Conduct Stringent Syringe Wash

The option to wash the syringe with a solution in the wash plate between transfers. Select the check box to perform the wash. Clear the check box to skip the wash.

Default: Number of Syringe Washes between Transfers

The number of Syringe Washes to be performed at the Wash Station, before adding liquid to the next sample plate. Range: 010 Default: 5

Number of Final Syringe Washes

The number of times to rinse syringes with deionized water. Range: 010 Default: 3

Property Description

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.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the protocol run.

To monitor the progress of the run, check the Status box.

When the run is finished, remove the labware from the deck.

WARNING To stop a run in an emergency, use the emergency stop pendant.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Syringe Test v2.0 User Guide

The Syringe Test v2.0 utility and the AssayMAP Syringe Test Kit enable verification of the integrity of the probes and syringes in the Bravo 96AM Head.

When to use the AssayMAP Syringe Test Kit As a part of routine hardware quality control to verify head functionality To verify the functioning of one or more syringes that produce suspicious results After a Bravo 96AM Head collision in which syringes may have been damaged After replacing any syringes in the Bravo 96AM Head

How it works The AssayMAP Syringe Test kit contains a rack of resin-free cartridges, labware, and a viscous dye solution. You place the resin-free cartridges and labware containing the dye solution on the Bravo deck at the specified locations, and then run the Syringe Test utility. The Syringe Test protocol does the following: Mounts the 96 resin-free cartridges on the syringes in the AssayMAP head. Places the cartridge tips in the syringe test solution. The syringes are quickly drawn

to a 225 L draw position. The speed of the aspiration is faster than the liquid can be drawn through the resin-free cartridges into the syringes, so a partial vacuum is formed in the syringes. The cartridges remain in the syringe test solution for 5 minutes to allow the test solution to be drawn into the syringes.

Ejects the cartridges, and then dispenses the dye solution into a 96-well flat bottom plate.

The absorbance of dye solution in each well of the plate is measured using a microplate reader. If a syringe is damaged or the seal between the syringe probes and the cartridges is compromised, air will be drawn into the syringes from the vacuum. The air leak will decrease the amount test solution drawn into the syringes and have a lower signal identifying the syringe as damaged.

Hardware

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

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Figure AssayMAP Bravo Platform components

CAUTION To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 L cartridges.

Note: The white wide-bore chimneys work for both 5-L and 25-L cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station or the later model 96 Channel Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Risers, 146 mm

7 STC controller

8 Peltier Thermal Station with custom plate nest

9 Thermal plate insert

10 Orbital Shaking Station with Control Unit

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Additional required equipment

Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

AssayMAP Syringe Test Kit components

One AssayMAP Syringe Test Kit provides enough materials and reagents to perform 20 tests of the 96 syringes in the Bravo 96AM head. The following table provides a description of the kit components.

Required equipment Description

Centrifuge Centrifuge with a microplate rotor capable of achieving 500 x g

Microplate reader Spectrophotometer capable of detecting absorbance at 425 nm in a 96-well microplate and generating a text (TXT) file of the data. Note: Although 425 nm is optimal, 405 nm is also acceptable.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

Component Description Part Number Quantity

Syringe Test Kit includes

G5496-60050 1

AssayMAP Resin- Free Cartridges

96 cartridges used for testing G5496-60009 1 rack

Test solution Aqueous colormetric solution with appropriate viscosity

5190-7471 3 bottle (250 mL each)

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom

Required reusable labware 201254-100 2

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Labware requirements

The Syringe Test requires the following labware: Open reservoir, (2 per test) to hold water (deck location 4) and diluted test solution

(deck location 7). 96-well microplate, (2 per test) for test sample collection and a centrifugation

balance plate (deck locations 8 and 9). The following figure shows the labware locations on the Bravo deck. The table lists the options for labware type by deck location.

Table Labware options by deck location

96 Greiner 655101 PS Clr Rnd Well Flat Btm

Required reusable labware 655101 2

Component Description Part Number Quantity

Labware options Manufacturer part number Deck location

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom

Agilent 201254-100 4, 7 (default)

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96- LP

4, 7

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 4, 7

96 V11 Manual Fill Reservoir Agilent G5498B#049 4, 7

96 Greiner 655101 PS Clr Rnd Well Flat Btm Greiner Bio-One 655101 8, 9 (default)

96 Nunc 269620, Flat Bottom, Polystyrene Thermo-Fisher 269620 8, 9

96 Corning 9017, Flat bottom, polystyrene, clear Corning 9017 8, 9

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Workflow

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Setting up the Syringe Test protocol

The following procedure specifies volume requirements for the default labware selections. If you choose different labware types from the list of options, you should adjust the volumes accordingly.

Step For this task... See...

1 Run the Startup protocol in the System Startup/Shutdown utility.

Setting up the Startup protocol on page 576

2 Set up the Syringe Test protocol. Setting up the Syringe Test protocol on page 553

3 Set up the AssayMAP Bravo deck for the syringe test.

Setting up the Syringe Test protocol on page 553

4 Run the Syringe Test. Starting the Syringe Test protocol run on page 555

5 Analyze the results. Analyzing the results on page 559

6 Verify defective syringes. Verifying defective syringes on page 565

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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To set up the protocol: 1 Open the Utility Library. Locate Syringe Test, and then click Utility.

The Syringe Test utility opens.

2 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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3 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

4 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Syringe Test protocol run on page 555.

To create or modify a method, proceed to step 5. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

5 In the Labware Table area, select the labware you are using for the protocol run. 6 Optional. To have the Centrifuge Balance Plate filled with 200 L water by the Bravo

Platform, select the Generate Balance Plate check box. 7 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Syringe Test protocol run

To start the Syringe Test protocol run: 1 Place the accessories or components on the AssayMAP Bravo deck.

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2 Ensure the following: The physical layout on the Bravo deck matches the Deck Layout image in the

form. The selections in the Labware Table reflect the labware you are using for the

protocol run. The labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

3 Dispense 80 mL of water into the reservoir at deck location 4. 4 Dispense 35 mL of the Test Solution into the reservoir at deck location 7.

Deck location

Accessory or component Purpose

1 96AM Wash Station or the later model 96 Channel Wash Station

Washes syringes and exterior cartridges

2 96AM Cartridge & Tip Seating Station

Ensures proper seating of the cartridges on the head

4 Reservoir, Seahorse 201254-100, PP (default)*

Holds water for the balance plate and dilution of test solution.

6 Cartridge rack with 96 resin-free cartridges

Holds test cartridges at beginning and end of protocol

7 Reservoir, Seahorse 201254-100, PP (default)*

Holds the 1X test solution

8 96 Greiner 655101 PS Clr Rnd Well Flat Btm (default)*

Microplate of same type as the collection plate for balancing in the centrifuge

9 96 Greiner 655101 PS Clr Rnd Well Flat Btm (default)*

Receives the test solution from the syringes and is used for the absorbance read.

* For other options, see Labware requirements on page 552.

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5 Click to start the run.

After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol.

WARNING To stop a run in an emergency, use the emergency stop pendant.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

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Automation movements during the Syringe Test run

This section describes the basic movements of the AssayMAP Bravo Platform during the Syringe Test protocol using the Default Method settings. Changing the selections or parameters will alter the movements.

Protocol step Head moves to location...

Action

Start protocol 1 Dispenses any liquid remaining in the syringes into the wash station between the chimneys.

1 Washes the syringes.

Cartridge transfer 6 Mounts the resin-free cartridges on the head.

2 Parks the cartridges in the seating station.

Water transfer 4 Aspirates 125 L of water.

9 Dispenses 100 L water.

1 Dispenses 25 L water.

4 Mixes 5 times, and then aspirates 200 L of water.

8 Dispenses 200 L of water.

Internal Cartridge Wash 4 Aspirates 150 L water.

2 Dispenses 10 L of water into the cartridge cups.

2 Mounts the cartridges on the head.

1 Dispenses 100 L of water through the cartridges into the wash station between the chimneys.

1 Washes the exterior tips of the cartridges.

2 Parks the cartridges in the seating station.

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Analyzing the results

How the data is analyzed The absorbance measurement of each well is compared to an expected reference value, which is determined from a statistical analysis of the data from all wells. Any syringe that has a corresponding value > 10% of the reference value fails the test.

To analyze the syringe test results: 1 After the run is completed, remove the collection microplate and the balance

microplate from the Bravo deck and spin them in a centrifuge at 500 x g for 1 min to remove any bubbles within the wells.

2 Set up the plate reader to:

Test Solution transfer 1 Dispenses 40 L of water into the wash station between the chimneys.

2 Aspirates a 25 L air gap.

2 Mounts the cartridges on the head.

7 Submerges the cartridges in Test Solution. Rapidly aspirates 225 L Test Solution and equilibrates for 5 min with the cartridge tips submerged.

2 Parks the cartridges in the seating station.

1 Dispenses 125 L of Test Solution into the wash station between the chimneys, and then washes the exterior of the syringe probes.

9 Dispenses 100 L of Test Solution and mixes it.

9 Dispenses 5 L of the air gap.

1 Dispenses remaining 20 L of the air gap.

1 Washes the syringes.

Cup wash 4 Aspirates 50 L of water.

2 Washes the cartridge cups and exercises the cartridges off task.

1 Dispenses the water into the wash station between the chimneys, and then does an external probe wash.

Internal Cartridge Wash 4 Aspirates 150 L water.

2 Mounts the cartridges on the head.

1 Dispenses the water into the wash station between the chimneys, and then washes the exterior of the cartridge tips.

6 Ejects the cartridges.

Final syringe wash 1 Washes the syringes.

Protocol step Head moves to location...

Action

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Detect absorbance at 425 nm Note: Although 425 nm is optimal, 405 nm is also acceptable.

Save the results to a file with a text (TXT) file format Note: Alternatively, you can save the data to a Microsoft Excel file (XLSX) and then use the copy and paste commands to transfer the data to the Analysis Tool.

3 Transfer the file to the computer running the Protein Sample Prep Workbench.

4 In the Syringe Test form, click to open the AssayMAP Syringe Test Analysis Tool. The AssayMAP Syringe Test Analysis Tool opens in Microsoft Excel.

IMPORTANT In Microsoft Excel, ensure that you enable content.

5 To choose the data for analysis: a Click Select data. b In the Data Selection Options dialog box, click Import New Data.

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c In the Import Text File dialog box, select the text (TXT) data file you want to analyze and click Import.

d Follow the instructions in the text import wizard: In the Text Import Wizard Step 1 of 3 dialog box, click Finish to accept the

default settings. In the Import Data dialog box, make sure Existing worksheet is selected

and then click OK.

e When the Select Data dialog box appears asking you to Select a continuous 8 row by 12 column range of cells to analyze, select the 96 cells you want to analyze and click OK. You must select 96 cells, otherwise you will receive a message requesting you to select 96 entries.

The data appears in the Analysis Tool window.

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6 Click Analyze. The Analysis Tool analyzes the imported data and displays the results in the Analysis - Plate View area.

Failed syringes are indicated by red cells. A list of the failed syringes is located below the analysis results.

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To verify that failures are defective syringes, go to Verifying defective syringes on page 565.

7 Save the results of the test: a Click Save Report. b Read the warning message that opens, and then click OK.

c In the Control Panel login window, type your VWorks user name and password, and click Log In.

d In the Save File dialog box, specify the file name and the storage location:

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Make sure the file name is unique to avoid creating multiple versions of the Syringe Test report with the same name, for example, YYYYMMDD_Syringe Test The default Shared Service storage location is VWorks Plus:

/VWorks Projects/VWorks/Experiments/Reports VWorks Standard:

C:\OLSS Projects\VWorks Projects\VWorks\Experiments/Reports Click Save.

8 Click OK when the uploaded successfully message appears.

Exporting a Syringe Test report

To create a copy of the report that you can open in Excel, use the following procedure to export the report.

To export a Syringe Test report:

1 In the navigation pane of the Syringe Test form, click to turn off full screen display so that the VWorks menu bar is visible.

2 In the VWorks window, click File > Export Misc File.

3 In the Open File dialog box, select the report (.csv file) and then click Open.

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4 In the Export Miscellaneous File dialog box, select the destination export location, and then click Save.

Verifying defective syringes

Use the following procedure to verify that failed test results indicate defective syringes and not a non-syringe-related problem. This procedure specifies volume requirements for the default labware selections. If you choose different labware types from the list of options, you should adjust the volumes accordingly.

Before you start Check the wash station carboys to ensure:

Source carboy has a sufficient supply of source water. Waste carboy has sufficient unused capacity to accept the waste water from

the run. Prime the wash station by running the Startup protocol. See Setting up the Startup

protocol on page 576. Rotate the rack of test cartridges at location 6 by 180 so that the cartridge that

was in the A1 position is now in the H12 position. Add 65 mL of water to the remainder of the liquid in the Water Reservoir at deck

location 4. Add 22 mL of Test Solution to the remainder of the liquid in the Test Solution

Reservoir at location 7.

Procedure

To verify defective syringes: 1 Rerun the Syringe Test utility protocol. See Starting the Syringe Test protocol run

on page 555. 2 Analyze the data. See Analyzing the results on page 559. 3 Compare the results to the first test:

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If a syringe fails both tests, the syringe is defective and should be replaced. See Replacing defective syringes on page 566 for repair options.

If a failing test result is not replicated, the syringe is acceptable for use. The initial failing result might have been the result of a non-syringe related problem. For example, a faulty cartridge will give a failing result on both the test and retest, but the location of the failure will track with the cartridge. This is why the cartridge rack is rotated by 180-degrees before the verification test.

Replacing defective syringes

To replace damaged syringes, do one of the following: Use the AssayMAP Syringe Replacement Kit (part number G5409-68002), and

follow the included instructions. Contact Agilent Technical Support to schedule a service call. If you continue to use the AssayMAP Bravo Platform while awaiting repair, do not use data obtained from the defective syringe.

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Syringe Wash v3.0 User Guide

The Syringe Wash v3.0 utility enables you to conduct up to 6 sequential syringe washes, using up to 6 different wash solutions (plus, water in the wash station). You select which buffer to use for each of the 6 syringe washes. The same wash buffer can be selected for multiple washes.

Before you start

Labware The following table provides a complete list of labware options and the corresponding deck locations.

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

IMPORTANT Make sure the 96AM Cartridge & Tip Seating Station at deck location 2 is empty. (The station should not contain cartridges or pipette tips.)

Figure Labware locations on the Bravo deck (top view)

Labware options (deck locations 38) Manufacturer part number*

1.2mL Deep-Well PolyPro Clear Plate ABgene AB-1127

96-Well U-Bottom PolyPro Clear Plate Greiner 650201

12-Column Low-Profile Reservoir Agilent 201280-100

8-Row Low-Profile PolyPro Reservoir Agilent 201282-100

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Reagents

An wash solution of 50 mM NaOH has been effective for washing the syringes between most protein and peptide sample preparation runs. The Syringe Wash utility provides up to 6 washes so that you can use multiple washes using solutions that cover different chemical spaces to address contaminates that are difficult to remove from the syringes.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom

Agilent 201254-100

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96-LP

96 V11 Manual Fill Reservoir Agilent G5498B#049

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Labware options (deck locations 38) Manufacturer part number*

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Syringe Wash utility.

To set up the protocol: 1 Open the Utility Library. 2 Locate Syringe Wash, and then click Utility.

The Syringe Wash utility opens.

3 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Syringe Wash run on page 572. To create or modify a method, proceed to step 6. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

6 In the Application Settings area, specify the following settings for the run: a Under Wash Settings, specify the number of Syringe Washes at the wash

station at the beginning and end of the run: Initial Syringe Washes. The number of standard Internal Syringe Washes

to be conducted using the wash station at deck location 1, before starting any of the syringe washes.

Final Syringe Washes. The number of standard Internal Syringe Washes to be conducted using the wash station at deck location 1, after completion of all the syringe washes.

b Set the following properties for each syringe wash. You can conduct up to six washes:

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Property Description Buffer Set the deck location of the Buffer reservoir for the

given syringe wash. The same deck location can be used for multiple syringe washes.

Defaults: Wash 1: Buffer 1 (deck location 4) Wash 2: Buffer 2 (deck location 5) Wash 3: Buffer 3 (deck location 6) Wash 4: Buffer 4 (deck location 7) Wash 5: Buffer 5 (deck location 8) Wash 6: Buffer 6 (deck location 9)

Wash Type The type of wash: NONE. Select this option to specify no wash.

If you select NONE for any of the 6 syringe washes, its corresponding syringe wash will be skipped.

Cycle in Reservoir. Select this option to conduct aspirate-and-dispense cycles in the designated wash buffer location. This option will not move the wash solution out of its original location.

Discard Waste Plate. Select this option to aspirate from the specified Wash Buffer reservoir and dispense into the Waste Plate at deck location 3.

IMPORTANT Ensure that the settings you specify will not overflow the capacity of the labware designated as the Waste Plate.

Discard Wash Station. Select this option to aspirate from the specified Wash Buffer reservoir and dispense into the wash station waste sump.

Default: Wash 1: Cycle in Reservoir Wash 2 Wash 6: None

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7 In the Labware Table, select that labware you are using: a Deck Location 3. Select the labware for the Waste plate. b Deck Location 49. Select the labware for the Buffer at each deck location that

you are using for this wash method. Note: The wash method can include up to six syringe washes.

8 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Syringe Wash run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form.

Volume (L) The volume to be aspirated into the syringes and dispensed for each wash cycle. Range: 0250 Default: 250

Cycles The number of aspirate-and-dispense cycles to be conducted for each syringe wash. Range: 060 Default: 5

Rinse after Each Cycle To wash the syringes at the wash station after each wash cycle in this wash, select the check box. Otherwise, clear the check box. Default: Not selected

Flow Rate (L/sec) The volumetric speed of the pipetting axis (w-axis) during each wash cycle. Range: 0300 Default: 200 Note: Although the AssayMAP flow rate is normally expressed in L/min, use L/sec on this form.

Wash Cycles The number of Internal Syringe Washes at the wash station to be conducted after all the wash cycles are completed for the given wash. Range: 010 Default: 0

Property Description

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Make sure the volume of solution in the labware is sufficient for the overage requirement plus the parameters selected on the form. For volume overage requirements for a given labware type, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench. Make sure the labware are properly seated on the Bravo deck.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

IMPORTANT Ensure that the volume parameter specified in the form will not result in the overflow of waste at deck location 3.

2 Click to start the run.

To monitor the progress of the run, check the Status box in the upper right corner of the form. When the run is finished, remove the labware from the deck.

WARNING To stop a run in an emergency, use the emergency stop pendant.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

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System Startup/Shutdown v3.0 User Guide

The System Startup/Shutdown utility consists of two protocols: Startup. Prepares the AssayMAP Bravo Platform for use by:

Initializing the device if this is the first time the Startup utility is run after powering up the AssayMAP Bravo Platform.

Dispensing any remaining liquid in the syringes to the wash station. Note: The w-axis (aspirate/dispense axis) is the up-and-down motion of the piston. The w-axis initialization results in emptying the syringes.

Priming the wash station lines. Washing the syringes the specified number of wash cycles. You should run Startup when you first start up the AssayMAP Bravo Platform, before running an AssayMAP Bravo application or utility if the Shutdown procedure was performed after the last AssayMAP run, or to return the AssayMAP Bravo to a known state.

Shutdown. Prepares the AssayMAP Bravo Platform for idle time by: Washing the syringes the specified number of wash cycles. Aspirating 200 L of Syringe Storage Liquid (water or a user-defined solution)

into the syringes. The Syringe Storage Liquid prevents residual reagents from forming salts and corroding the syringe seals.

You should run Shutdown after every application protocol run, if the AssayMAP Bravo Platform will not be in use for at least 1 hour, and before turning off the device.

Before you run the Startup protocol

Before running the Startup protocol: 1 Check the liquid levels of the source and waste bottles for the wash station. Fill the

source bottle and empty the waste bottle, as required. Verify that the flip-top cover on each pump head is closed and that the tubing connections are secure.

Figure Wash station fill line (A) and empty line (B)

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2 Turn on the Pump Module and Peltier Thermal Station Controller. 3 Ensure the following items are in position on the AssayMAP Bravo deck:

96AM Wash Station or the later model 96 Channel Wash Station at deck location 1

Empty 96AM Cartridge & Tip Seating Station at deck location 2 4 Turn on the AssayMAP Bravo Platform. 5 Open the System Startup/Shutdown utility.

Before you run the Shutdown protocol

Before running the Shutdown protocol: 1 If more rigorous syringe washing is desired, run the Syringe Wash utility before

running the Shutdown protocol. For instructions, see Syringe Wash v3.0 User Guide on page 567.

2 Check the liquid levels of the source and waste bottles for the wash station. Fill the source bottle and empty the waste bottle, as required. Verify that the tubing connections are secure.

3 Ensure the following items are in position on the AssayMAP Bravo deck: 96AM Wash Station or the later model 96 Channel Wash Station at deck

location 1 Empty 96AM Cartridge & Tip Seating Station at deck location 2 Optional. Acceptable reservoir containing an appropriate Syringe Storage

Liquid at deck location 7 4 Open the System Startup/Shutdown utility.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Opening the System Startup/Shutdown utility

To open the Startup/Shutdown utility: In the Utility Library, locate the System Startup/Shutdown utility, and then click Utility.

The System Startup/Shutdown utility opens.

Setting up the Startup protocol

To set up the Startup protocol: 1 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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2 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

3 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Startup run on page 578. To create or modify a method, proceed to step 4. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

4 In the Startup Options area of the form, enter the values for the following properties:

5 To save the method:

a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Property Description Number of Syringe Wash Cycles

Specifies the number of syringe wash cycles that will be conducted at the wash station at deck location 1. Default: 3 Range: 010

Wash Station Prime Duration (sec)

Specifies how long (seconds) to run the wash station pumps in order to prime the tubing lines and wash station chimneys. If the tubing lines are dry, set this value to 60 seconds. Default: 10 Range: 0300

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Starting the Startup run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories are at the assigned deck locations, as shown in the

Deck Layout image of the form.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click .

If this is the first time the Startup utility has been run after powering up the AssayMAP Bravo Platform, the device initialization process begins. Proceed to step 3.

If the platform is already initialized, skip to step 5. 3 When the following message appears, click Ignore and Continue, leaving device in

current state to proceed to initialize the grippers.

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4 When the following message appears, click Ignore and Continue, leaving device in current state to delay initializing the w-axis. The w-axis will automatically initialize in a later step when the Bravo 96AM Head is positioned over the wash station at deck location 1.

When the initialization process is finished, the orange lights on the AssayMAP Bravo Platform light panel flicker briefly and then begin to flash. The Bravo 96AM Head does the following: Moves to deck location 2 and parks any cartridges remaining on the head.

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Moves to the wash station at deck location 1 and dispenses any liquid remaining in the syringes.

Washes the syringes the specified number of times. The priming step begins after the syringe wash.

5 During the priming step, ensure that fluid flows out of the top of each chimney. Check for any damaged chimneys in the wash station. Click Continue to complete the Startup protocol.

When all the chimneys are flowing evenly, the device is ready for operation.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Setting up the Shutdown protocol

To set up the Shutdown protocol: 1 If applicable, click Select Experiment ID.

The Experiments Editor opens.

Troubleshooting problems

Probable cause and solution

Fluid does not flow out of a chimney.

A trapped air bubble can block the fluid flow out of a chimney.

To clear the bubble: 1 Push the air out of a 1000 L pipette with a pipette tip

attached. Form a seal between the top of the problematic chimney and the 1000-L pipette tip, and then draw the air and then water through the chimney at a medium speed.

2 Run the Startup protocol again to determine if the bubble was successfully removed.

3 Repeat if necessary. Chimney deformity or irregularity

Chimney irregularities can result from a collision between the head and the wash station. For additional troubleshooting guidelines or to replace damaged chimneys, see the 96 Channel Wash Station Maintenance Guide.

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2 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

3 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

To run the selected method, go to Starting the Shutdown run on page 582. To create or modify a method, proceed to step 4.

VWorks Plus. Administrator or technician privileges are required to create and modify methods.

4 In the Shutdown Options area of the form, enter the values for the following properties:

5 To save the method:

Property Description Number of Syringe Wash Cycles

Specifies the number of syringe wash cycles to conduct at the wash station at deck location 1. Default: 3 Range: 010

Syringe Storage Liquid Source

Determines the location from which to aspirate the syringe storage liquid. The options are: 96AM Wash Station. The liquid is aspirated from the

chimneys of the wash station at deck location 1. Syringe Storage Liquid. The liquid is aspirated from

the labware that you specified at deck location 7. For this option, ensure that you: Select the labware type for deck location 7 in the

Labware Table area. Provide the minimal volume overage required for

the labware type being used, plus 200 L per syringe. For the labware overage requirements, see the Labware Reference Guide (Workbench Literature Library page).

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a Click .

b In the Save File As dialog box, type the file name and click Save. VWorks Plus. You must save the method before you can run it.

Starting the Shutdown run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the Shutdown run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click . The shutdown process begins.

3 Observe the AssayMAP Bravo head as it does the following: Moves to deck location 2 and parks any cartridges remaining on the head. Moves to the wash station at deck location 1 and dispenses any liquid

remaining in the syringes. Washes the syringes the specified number of times. Aspirates the 200 L of Syringe Storage Solution into the syringes from the

specified location. 4 When the protocol finishes:

a Remove all labware from the AssayMAP Bravo deck. b Discard the excess Syringe Storage Liquid, if necessary. c Empty the waste bottle.

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WARNING Make sure you discard the chemical waste according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

5 If the AssayMAP Bravo Platform will remain unused overnight: a Exit the Protein Sample Prep Workbench. b Shut down and turn off the computer. c Turn off the Bravo Platform. d Turn off the accessories. e At the Pump Module, lift the flip-top cover on each pump head to prevent

flattening the tubing and to help maximize the tubing life.

Automation movements Startup protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Startup protocol using the default protocol method. Changing the selections or parameters will alter the movements.

Automation movements Shutdown protocol

This section describes the basic movements of the AssayMAP Bravo Platform during the Shutdown protocol using the default method settings. Changing the selections or parameters will alter the movements.

Head moves to deck location...

Action

2 Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

1 Washes the syringes the specified number of times.

2 Moves the head to a safe height above deck location 2 to enable user observation of the wash station at deck location 1.

1 Primes the wash line and fills the wash station with liquid from the wash buffer reservoir for the specified duration.

1 Moves the head to a safe height above deck location 1.

Protocol steps

Head moves to deck location...

Action

2 Parks any cartridges that may have been mounted on the head from a protocol that had been previously aborted.

1 Dispenses any liquid remaining in the syringes into the wash station.

1 Washes the syringes the specified number of times.

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1 Fills the wash station chimneys with DI water.

1 or

7

Aspirates 200 L of DI water from the wash station into the syringes.

Aspirates 200 L of Syringe Storage Liquid from the reservoir at deck location 7 into the syringes.

1 Moves the head to a safe height above deck location 1.

Protocol steps

Head moves to deck location...

Action

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This chapter contains the following topics: Utility description on page 586 Before you start on page 586 Setting up a Normalization method on page 590 Preparing the diluent and samples on page 609 Running the protocol on page 610 Assay development guidelines on page 615

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Utility description

Normalization v2.0. This utility enables you to convert a sample plate containing samples of various concentrations into a sample plate containing samples of a uniform concentration. The utility cycles through each sample, one at a time, combining a calculated volume of each sample with an appropriate volume of diluent.

Before you start

This topic lists the required hardware, software, labware, and solutions for running the Normalization protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The AssayMAP Bravo Platform is required for running the AssayMAP protocols. The following figure and table describe the platform components.

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Figure AssayMAP Bravo Platform components

Optional equipment. The following equipment is recommended when preparing the samples and reagents: Microplate centrifuge, such as the Agilent VSpin Microplate Centrifuge or

equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Bravo Risers, 14.6 cm

7 Peltier Thermal Station with STC controller

8 Plate Risers, 2.84 cm (two)

9 Orbital Shaking Station with Control Unit

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For an overview of the software components, see Overview of software architecture on page 15.

Labware

The Normalization protocol requires the following labware: Full tip box of 96 250-L pipette tips, Agilent 19477-002 Labware for the diluent reservoir, initial samples plate, and normalized samples

plate The following table provides a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Using different pipette tips, such as filtered tips, will result in equipment damage. Ensure that you use the standard 250-L pipette tip (Agilent 19477-002).

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Accessory and labware locations on the Bravo deck (top view)

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

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The unusable volume (also known as dead volume) is the volume that cannot be reliably aspirated from the microplate well due to pipetting limitations that arise from differences in well-bottom geometry. The following table provides some general guidelines on how different microplate geometries can affect this value.

Table Guidelines on how microplate geometry can affect volume requirements

For volume overage recommendations by labware type, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Labware Manufacturer part number*

Deck location options

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom ABgene AB-1127 2, 6

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 2, 6

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 2, 6

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 2, 6

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 2, 6

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 2, 6

96 Costar 3363, PP Conical Bottom Corning Costar 3363 2, 6

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 2, 6

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 4

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 4

96 V11 Manual Fill Reservoir Agilent G5498B#049 4

Reservoir, Axygen Scientific RES-SW96-LP, 86mL pyramid bottom

Axygen Scientific RES-SW96-LP

4

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Well-bottom geometry Examples Unusable volume expectations

Flat bottom Greiner 675801 Generally, the worst plates for minimizing dead volume. Liquids are attracted to the intersection of the plate bottom and side walls.

Round (U) bottom Greiner 650201, Greiner 650207 Generally, good options for minimizing dead volume. A round bottom forces liquid to collect in the center of the well at a medium rate.

V-bottom and PCR V-Bottom: Greiner 651201, Corning Costar 3363

PCR Plate: Eppendorf 30129300, Greiner 652270

Generally, the best plates for minimizing dead volume. Steep side walls force liquid to collect in the center of the well, making it easy to access.

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Solutions

The Normalization utility requires the following solutions: Diluent to adjust the sample concentration Purified water for the wash station reservoir (carboy)

Setting up a Normalization method

The Method Setup Tool is a Microsoft Excel-based tool that steps you through the process of creating and saving a method file that contains all the information required to run the Normalization protocol on the AssayMAP Bravo Platform. This tool uses formulas to calculate volume requirements for the samples and diluent based on your input. Note: When you select a method in the Normalization utility, the form displays the corresponding labware selections and diluent preparation instructions.

Figure Normalization Method Setup Tool

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Before you start

Ensure that you have a Microsoft Excel file or a delimited text file (.csv or .txt) that contains the sample concentration values to be imported. The file should contain an 8-by-12 array of values, where Each row of the 8-by-12 array represents a row in the 96-well microplate. Each column of the 8-by-12 array represents a column in the 96-well microplate. An empty cell or a value of 0 is acceptable.

Figure Example of an Excel file with an 8-by-12 grid numbers

Figure Example of a csv file with an 8-by-12 grid numbers

Opening the Method Setup Tool

You can open the tool from the Utility Library or from the Normalization form.

To open the Method Setup Tool: 1 In the Utility Library, locate the Normalization banner.

2 Click one of the following buttons: Method Setup Tool. Microsoft Excel starts and displays the Method Setup Tool. Utility. The Normalization utility opens.

In the navigation pane, click Method Setup Tool. Microsoft Excel starts and displays the Method Setup Tool.

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IMPORTANT In Microsoft Excel, ensure that you enable content.

Overview of steps in Method Setup Tool

The Method Setup Tool has 13 distinct steps. The following table provides an overview of the steps.

Table Overview of steps in Method Setup Tool

Step Description

Defining Initial Samples plate (steps 14)

1 Import concentrations

Click Import Sample Concentrations, and then import the values for the known concentrations in your sample plate from a file (.xls, .xlsx, .csv, or .txt) or enter the values manually.

2 Define Concentration Units

Enter the concentration units in the green box. Note: This input is only a reminder. It has no impact on the subsequent calculations.

3 Select Initial Samples Plate

Click the green Sample Plate box, and then select the labware from the list. The setup tool uses the value that appears in the Unusable Volume box (also known as dead volume) to calculate the usable sample volume. You can change the unusable volume manually, if desired, as the default value is a conservative estimate.

4 Enter Initial Sample Volume

Type the initial sample volume in the green (L) box, and then select the array of wells for which this value is correct in the Usable Sample Volumes plate grid. The setup tool automatically enters the usable sample volume (that is, the initial sample volume minus the unusable volume) in the Usable Sample Volumes plate grid. Repeat step 4 until you have specified the volume for all the wells in the Sample plate that contain a sample.

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Defining normalization targets (steps 56)

5 Enter the Target Concentrations

Type the target concentration in the green box, click Select Wells to Fill, and then select the array of wells to be filled in the Target Sample Concentrations plate grid. Repeat step 5 until you have specified the concentrations for all the wells.

6 Enter the Target Volume

Type a value in the green (L) box that you want to apply to multiple wells of the microplate, and then click Select Wells to Fill. Select the array of wells to be filled in the Target Final Volumes plate grid. Repeat step 6 until you have specified the volumes for all the wells.

Calculating results and dealing with exceptions (steps 78)

7 Calculate Normalization Volumes

Click Calculate Volumes. The calculated volumes (L) display in the Diluent Volumes to Use and the Sample Volumes to Use plate grids.

8 Manage Calculation Exceptions

Click Check Exceptions. In the Manage Exceptions dialog box, select the option that is appropriate for each sample that is flagged as problematic. The setup tool wizard guides you through the process.

Finalizing Normalization method setup (steps 913)

9 Define Remaining Labware Types

Click the green Normalized Plate box, and then select the labware from the list. The Maximum Volume (L per well or channel) for the selected plate is automatically displayed. Note: The final normalized sample volume must be equal to or less than the well volume of the selected plate type, or the Maximum Volume (L per well or channel) cell will turn red. Click the green Diluent Reservoir box, and then select the labware from the list.

10 Select the Number of Mix Cycles

Click the green mix cycles box, and then select the value from the list. Note: The number of mix cycles is dependent on the volume, viscosity, and size of molecules in the solution being mixed.

11 Input Evaporation Correction Factor

Type a value in the green (%) box for the correction factor, and then click Apply Correction Factor. The setup tool automatically adjusts the values in the Final Diluent Volumes and Final Sample Volumes plate grids.

12 Check Volume Required for Diluent Reservoir

Review the Diluent required for Diluent Reservoir area at the bottom of the worksheet. If a red highlight appears on the Total to fill Reservoir value, the diluent volume plus required overage is more than the selected labware can hold. In this case, select a different labware option for the Diluent Reservoir in step 9. Make a note of the Total to fill Reservoir volume. You will use this value to prepare the diluent reservoir for the Normalization run. This volume includes the required overage.

13 Create a Normalization Method File

Click Create Method File. After you save the method, the Method Setup Tool closes. When you select the method in the Normalization utility, the form displays the corresponding labware selections and diluent preparation instructions. Follow the instructions that appear in the form to prepare the diluent and sample plates.

Step Description

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Step 1) Import Concentrations

To import the values for the known concentrations into the initial samples plate, use one of the following procedures: Importing values from an Excel file Importing values from a text file Note: Empty cells or cells with a value of 0 are acceptable.

Importing values from an Excel file

To import the values from an Excel file (.xls or .xlsx): 1 To clear any data from the Normalization Method Setup Tool, click Clear All (upper

right corner). 2 Click Import Sample Concentrations. The Import Initial Sample Concentrations

dialog box opens.

3 Click Import concentrations from an Excel file (.xls or .xlsx). 4 In the Open Excel File dialog box, select the file, and then click Open.

The selected Excel file opens and the Select Data dialog box opens. 5 In the Excel file, select the 8-row by 12-column array of values to be imported, and

then click OK in the Select Data dialog box. Note: When selecting data from an Excel sheet, the selected range must be an 8-row by 12-column array of values that correspond to the well positions of the samples.

The Excel file closes and the imported values appear in the Initial Sample Concentrations area.

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Importing values from a text file

To import the values from a text file (.csv or .txt): 1 Click Import Sample Concentrations. The Import Initial Sample Concentrations

dialog box opens.

2 Click Import concentrations from a text file (.csv or .txt). 3 In the Import Text File dialog box, select the file and click Import. 4 In the Excel Text Import Wizard that appears, follow the instructions to specify the

type of delimited file.

5 In the Import Data dialog box, select Existing worksheet, and then click OK.

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The content of the imported file appears in the Restricted Import worksheet of the Method Setup Tool, and the Select Data dialog box opens.

6 In the imported worksheet, select the 8-by-12 array of values, and then click OK in the Select Data dialog box. The selected values appear in the Initial Sample Concentrations area.

Step 2) Define Concentration Units

To define the concentration units: In the green box next to Step 2) Define Concentration Units, verify that the unit of measure for the sample concentration is correct. If necessary, type the correct units.

This input is only a reminder. It has no impact on the subsequent data or volume calculations.

IMPORTANT Ensure this unit of measure is constant throughout the setup process. The tool does not adjust calculations to accommodate the use of different units for the initial and target concentrations.

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Step 3) Select Initial Sample Plate

To select the initial Sample plate: 1 Click the Sample Plate box, and then select the labware from the list.

2 Notice the value that automatically appears in the Unusable Volume box. To calculate the usable volume, the calculator will subtract this value from the value in Step 4) Initial Sample Volume. The unusable volume (also known as dead volume) is the volume that cannot be reliably aspirated from the microplate well due to pipetting limitations that arise from differences in well-bottom geometry. The default value for Unusable Volume is a recommended setting that is based on extensive testing with the labware. If necessary, you may change the value, for example if the value is not appropriate for the sample liquid.

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

For a list of recommended values for each allowable labware type, see Labware on page 588. Note: The Labware Reference Guide also presents labware-specific pipetting overage and maximum well capacity. You can find this guide in the Literature Library page of the Protein Sample Prep Workbench.

Step 4) Enter Initial Sample Volume

Use the following procedure to define a volume of sample that is initially present in each well of the Sample plate.

To enter the initial Sample volume: 1 In the green (L) box, type the initial sample volume. 2 Click Select Wells to Fill.

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3 When the Initial Sample Volume dialog box opens: a Select the array of wells to be filled in the Usable Sample Volumes plate grid of

the worksheet. b Click OK in the Initial Sample Volume dialog box to populate the selected wells

of the plate grid.

Note: The values that populate the plate grid represent the volume of the Initial Sample Volume that is usable. This value is the difference between the value you entered in the green box for Initial Sample Volume and the value in the Step 3) Unusable Volume box. If you want to clear all values from the Usable Sample Volumes plate grid, click Clear.

4 Repeat step 1 to step 3 until you have specified the volume for all the wells in the Sample plate that contain a sample.

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Step 5) Enter the Target Concentrations

IMPORTANT Make sure you consider what is possible given the Initial Sample Concentrations and Usable Sample Volumes from Step 1) and Step 4), respectively. Although you may enter values that are not possible or practical given the Initial Sample Concentration and Usable Sample Volume, the tool will flag such values as exceptions when calculating the normalization volumes. You will specify how to handle each exception in a subsequent step.

Use the following procedure to specify the desired concentrations after the samples are normalized.

To enter the target concentrations: 1 In the green box, type the concentration. 2 Click Select Wells to Fill.

3 When the Target Sample Concentration dialog box opens:

a Select the array of wells to be filled in the Target Sample Concentrations plate grid of the worksheet.

b Click OK in the Target Sample Concentration dialog box to populate the selected wells of the plate grid.

Note: If you want to clear all values from the Target Sample Concentrations plate grid, click Clear.

4 Repeat step 1 to step 3 until you have specified the concentrations for all the wells.

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Step 6) Enter the Target Volume

IMPORTANT Make sure you consider what is possible given the previous entries. Although you may enter values that are not possible or practical given the Initial Sample Concentration, Usable Sample Volume, or Target Concentration, the tool will flag such values as exceptions when calculating the normalization volumes. You will specify how to handle each exception in a subsequent step.

Use the following procedure to specify the desired volumes after the samples are normalized.

To enter the target volume: 1 In the green (L) box, type a value that you want to apply to multiple wells of the

microplate. 2 Click Select Wells to Fill.

3 When the Target Final Volume dialog box appears:

a Select the array of wells to be filled in the Target Final Volumes plate grid of the worksheet.

b Click OK in the Target Final Volume dialog box to populate the selected wells in the plate grid.

Note: If you want to clear all values from the Target Final Volumes plate grid, click Clear.

4 Repeat step 1 to step 3 until you have specified the volumes for all the wells. Note: If the next step in the workflow involves an AssayMAP application, all the samples should be at the same target volume.

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Step 7) Calculate Normalization Volumes

To calculate the normalization volumes: Click Calculate Volumes. An algorithm uses the information provided in the previous steps to calculate the sample and diluent volumes required to achieve the specified normalization targets. The calculated volumes (L) are displayed in the following areas: Diluent Volumes to Use plate grid Sample Volumes to Use plate grid

Any wells with red highlights are considered exceptions, for which the calculated sample or diluent volume values cannot be achieved because the volumes required are either larger than the volumes available in the Sample plate or smaller than practical for the AssayMAP Bravo Platform to transfer with high accuracy.

Step 8) Manage Calculation Exceptions

IMPORTANT You must click Check Exceptions in order to proceed, even if there are no red highlighted wells in the plate grid. The setup tool also performs other important conformity checks at this time.

In this step, you decide how to address each of the exceptions that are indicated by red highlighted wells in the plate grid.

To manage the exceptions: 1 Click Check Exceptions. The Manage Exceptions dialog box opens. The descriptions

change in the Status and the Selecting Do Best Will areas depending on the conditions that caused the exception for the selected sample. The following figure shows an example.

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Figure Example of the Manage Exceptions dialog box

2 Select the option that is appropriate for your samples. The options are as follows:

Option Description Do Best The resulting actions for using this option depend on the

conditions that caused the exception. An explanation of the action for the selected sample is displayed in the Selecting Do Best Will area. For descriptions by the type of exception, see the following section, Do Best option and types of exceptions. In general, Do Best adjusts the calculated sample and diluent volumes to get as close to the target concentration as possible while maintaining the target volume. The tool gives a higher priority to the target volume than the target concentration, assuming that the Normalized plate will be used in another AssayMAP Bravo run, and the well volume differences across the plate can lead to unpredictable assay results. The values from the calculations appear in the Expected Concentrations after Normalization plate grid at the bottom of the worksheet. You may apply Do Best in the following ways: Click Do Best for each exception, sample by sample. Select Apply Do Best or Skip Sample selection to all

samples, and then click Do Best. The action is performed on all the remaining exceptions.

After addressing all the exceptions, click Close to close the dialog box.

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Do Best option and types of exceptions If you select the Do Best option, the resulting Bravo actions vary depending on the conditions that caused the exception. The following four conditions cause exceptions:

1 The initial concentration value for a sample is lower than the target concentration for that sample.

2 The usable sample volume value is insufficient to cover the volume required to meet the normalization target concentration or target volume.

3 The calculated sample volume is less than 5 L, which is the minimum practical volume at which high accuracy and precision (5% CV, 10% Relative Inaccuracy) can be achieved when using the AssayMAP Bravo Platform.

4 The initial concentration for a sample is lower than the target concentration for that sample, and the usable volume in the initial Sample plate is not sufficient to allow for the entire sample to be transferred directly into the Normalized plate.

The following table describes the status messages and Do Best behaviors for each of the four exceptions.

Skip Sample This option skips the normalization process for the selected sample. If selected, nothing will be transferred into the Normalized plate at the specified well location. You may apply Skip Sample in the following ways: Click Skip Sample for each exception, sample by

sample. Select Apply Do Best or Skip Sample selection to all

samples, and then click Skip Sample. The action is performed on all the remaining exceptions.

After addressing all the exceptions, click Close to close the dialog box. Note: No pipette tip is used for blank wells or skipped wells.

Cancel Click Cancel to exit the Manage Exceptions dialog box if: No exceptions remain and you want to move on to

the final steps of defining the normalization method.

You want to return to an earlier step in the method setup process to make changes.

Apply Do Best or Skip Sample selection to all samples

The option to perform the Do Best or Skip Sample action on all the remaining wells. Select the Apply Do Best or Skip Sample selection to all samples check box, and then click Do Best or Skip Sample, as applicable. After addressing all the exceptions, click Close to close the dialog box.

Option Description

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As the exceptions are managed: If Do Best was selected, the new values calculated for the diluent and sample

volume are highlighted in the Final Diluent Volume and Final Sample Volume plate grids.

If Skip Sample was selected, the word Skip appears as the value for both the Final Diluent Volume and the Final Sample Volume.

IMPORTANT If you have exceptions, make sure you review the Expected Concentrations after Normalization values at the bottom of the worksheet before you create the method file.

Step 9) Define Remaining Labware Types

To specify labware for the remaining deck locations: 1 Click the Normalized Plate green box, and then select the labware from the list.

Note: This plate will be placed on a Bravo Plate Riser at deck location 6, which is where the final normalized samples will be at the end of the run.

2 Notice the value that automatically displays in the Maximum Volume (L per well or channel) box.

Exception Status message Do Best behavior

1 Initial concentration is below the target concentration.

No dilution will take place. Only sample will be transferred to the Normalized plate, which will cause the sample to have a concentration that is lower than the specified target concentration.

2 Sample volume required is more than is available in the Initial Sample plate.

The deficit in sample volume will be substituted with diluent, causing the concentration to be lower than expected, but ensure that the target volume is still met.

3 Sample volume required is less than 5 L. Five microliters is the minimum volume that can be transferred with a high degree of accuracy.

The AssayMAP Bravo Platform will attempt to perform the normalization as defined, by pipetting less than 5 L of sample.

4 Initial concentration is below the target concentration and the initial volume is less than the target volume.

The deficit in sample volume will be substituted with diluent, causing the concentration to be lower than expected, but ensure that the target volume is still met.

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IMPORTANT If this value has a red highlight, the selected labware cannot accommodate the volume specified in Step 6) Target Volume.

3 Click the Diluent Reservoir green box, and then select the labware from the list. Note: This reservoir will be placed at deck location 4 without a Bravo Plate Riser. The reservoir will supply diluent for the normalization process.

4 Notice the corresponding value in the Maximum Volume (L per well or channel) box. This value minus the dead volume associated with the selected plate type is the volume that the Bravo 96AM Head can access in the selected labware. The Bravo 96AM Head uses a single pipette tip mounted on probe A12 to aspirate the volume. For example, in a 12-column reservoir, the accessible volume is only the rightmost column, and in an 8-row reservoir, the accessible volume is only the topmost row.

Figure Upside down view of AssayMAP head showing probe A12

Step 10) Select the Number of Mix Cycles

In this step you select the number of mix cycles to perform after all the samples and diluent are added in the Normalized plate. The Bravo 96AM Head presses on the full selection of used pipette tips from deck location 3, and then mixes all the wells at the same time. The mixing volume is 75% of the cumulative plate volume, up to the maximum capacity of the pipetting method, where

Capacity for 250 L pipette tips = 140 L Normalization methods for large volumes may require a greater number of mix cycles, which you should determine empirically.

To select the number of mix cycles: Click the mix cycles box, and then select the value from the list.

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Step 11) Input Evaporation Correction Factor

In general, a Normalization protocol run of 96 samples on the AssayMAP Bravo Platform requires approximately 110 minutes. During this time, the samples can evaporate, especially by the time the 96th sample is transferred. The optional Evaporation Correction Factor helps to compensate for the sample evaporation. The evaporation rate will vary depending on the sample solution. You should empirically determine the correction factor using test samples. See Assay development guidelines for a detailed explanation of when to use this factor and how to calculate an appropriate value to use.

To account for sample evaporation: 1 In the Step 11) green box, type a value (%) for the correction factor, and then click

Apply Correction Factor. The tool automatically adjusts the values in the Final Diluent Volumes and Final Sample Volumes plate grids. The button label changes to Undo Correction Factor. Note: If you want to undo the change, click Undo Correction Factor.

Step 12) Check Volume Required for Diluent Reservoir below and Adjust If Necessary

CAUTION A small volume excess is required in all labware types to ensure proper volume transfer.

To verify that the Diluent reservoir has sufficient volume: 1 Review the Diluent required for Diluent Reservoir area at the bottom of the

worksheet. The Method Setup Tool calculates the total fill volume for the Diluent reservoir based on the calculated Final Diluent Volumes and the recommended excess volume for the selected labware type. Note: For a list of recommended values for each allowable labware type, see Labware on page 588.

2 If a red highlight appears on the Total to fill Reservoir value, the diluent volume required is more than the selected labware can hold. In this case, select a different labware option for the Diluent Reservoir in Step 9).

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Note: If you are using a 12-column reservoir or an 8-row reservoir, the Total to fill Reservoir value represents the volume in the rightmost column or topmost row, respectively.

3 Make a note of the Total to fill Reservoir volume. You will use this value to prepare the diluent reservoir for the normalization run.

Step 13) Create a Normalization Method File

Note: If you manually type the values into the plate grids of the Normalization Method Setup Tool, you may have unexpected results.

To create the Normalization method file:

1 Click .

2 In the login window, type your VWorks user name and password, and click Log In.

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3 In the Save File dialog box, specify the file name and the storage location, and then click Save.

VWorks Plus default storage location: /VWorks Projects/VWorks/AM Methods/AM Normalization v3.0

VWorks Standard default storage location: C:\OLSS Projects\VWorks Projects\VWorks\AM Methods\AM Normalization v3.0

Note: The method file is stored as a record of interest (ROI) in an archive (.csv.roiZip). VWorks cannot load files (.roiZip extension) that have been modified or renamed outside of the Protein Sample Prep Workbench or VWorks software.

4 Click OK when the uploaded successfully message appears. The Method Setup Tool closes.

Next steps: If you want to view the method in Microsoft Excel, see Exporting and importing AssayMAP methods on page 17 If you are ready to prepare the serial dilution plates, see Preparing the diluent and samples on page 609.

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Preparing the diluent and samples

Before you start

If you have not already done so, display the methods labware selections and diluent and sample preparation instructions by doing one of the following: Open the Normalization utility and select the method. For details, see Setting up

the protocol on page 611. Export the method and view it in Microsoft Excel. For details, see Exporting and

importing AssayMAP methods on page 17.

IMPORTANT To minimize evaporation, fill the Sample and Diluent plates immediately before run time or keep them covered until you run the protocol.

CAUTION Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the selections in the method exactly match the physical labware present on the Bravo deck.

Preparing the Diluent reservoir

To prepare the Diluent reservoir: Prepare the Diluent plate by putting the volume calculated by the Normalization Method Setup Tool in position A12. All the diluent will be aspirated from this location. Note: If you are using a 12-column reservoir or an 8-row reservoir, the Total to fill Reservoir value represents the volume in the rightmost column or topmost row, respectively.

Transferring samples to the microplate

To transfer the samples to the microplate: 1 Ensure that the labware type for the initial samples plate matches the labware

specified in the Normalization Method Setup Tool. 2 Run the Reagent Transfer utility or Reformatting utility to transfer the samples. For

instructions, see one of the following: Reagent Transfer v3.0 User Guide on page 525 Reformatting v3.0 User Guide on page 623 Ensure that the volume and well positions are as specified in the Normalization Method Setup Tool.

3 If necessary, centrifuge the sample labware to remove bubbles.

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Running the protocol

The Normalization protocol does the following: Washes and dries the syringes. Transfers diluent and sample to the normalized plate. Mixes all the samples in the normalized plate.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the Normalization Setup Method. See Setting up a Normalization method

on page 590. Display the methods labware selections and diluent and sample preparation

instructions by doing one of the following: Open the Normalization utility and select the method. For details, see Setting

up the protocol on page 611.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Export the method and view it in Microsoft Excel. For details, see Exporting and importing AssayMAP methods on page 17.

Prepare the diluent and samples. See Preparing the diluent and samples on page 609.

If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

Run the Startup protocol to prepare the AssayMAP Bravo Platform for the run. See the System Startup/Shutdown v3.0 User Guide utility.

Setting up the protocol

Before starting the protocol, make sure the appropriate selections and values are specified in the Normalization utility.

To set up the protocol: 1 Open the Utility Library. 2 Locate Normalization, and then click Utility.

The Normalization utility opens.

3 If applicable, click Select Experiment ID.

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The Experiments Editor opens.

4 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

5 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

The form displays the method labware selections and diluent preparation instructions. To run the selected method, go to Starting the protocol run on page 613. To create or modify a method, see Setting up a Normalization method on

page 590. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. You must use pipette tips for the mock run.

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Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form. The Bravo Plate Risers are securely in place at deck locations 2 and 6. The

initial samples plate is seated on the Bravo Plate Riser at deck location 2. The normalized samples plate is seated on the Bravo Plate Riser at deck location 6.

The Diluent reservoir is at deck location 4. The tip box full of fresh pipette tips is at deck location 3 and the empty 96AM

Cartridge & Tip Seating Station is at deck location 5. Note: A full tip box is required for each run even if the run uses less than 96 pipette tips. Note: The protocol will transfer the pipette tips to the seating station.

CAUTION To prevent a potential collision, ensure that no thermal plate insert is on the Peltier Thermal Station installed at deck location 4.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

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Note: After the protocol run starts, you can walk away from the AssayMAP Bravo Platform for the duration of the protocol. In general, 96 samples requires approximately 110 minutes.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

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a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck match

the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines

This topic provides guidelines for adjusting the protocol, and notes about the utility. Note: See the Labware Reference Guide for labware-specific maximum well capacity and other details. You can find this guide in the Literature Library page of the Protein Sample Prep Workbench.

Correcting for loss due to evaporation

Evaporation can cause problems for any normalization process, whether conducted manually or using automation. The time difference between the normalization of the first sample (A1) and the last sample (H12), leads to progressively increasing atmospheric exposure across the samples. This causes a gradual increase in the mass of sample that is transferred into the normalized sample plate, which leads to higher than expected concentrations for samples with long exposure times. The following figure shows the order in which the Normalization utility normalizes the samples. The

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utility starts with column 1 on the left and finishes with column 12 on the right. Without any evaporation correction, you would expect to see the normalized concentrations increase across the plate from left to right.

Figure Run order: the sequence in which samples are normalized

Step 11 of the Normalization Method Setup Tool contains an optional Evaporation Correction Factor to correct for this problem. The tool applies the factor to the calculated normalization volumes. The factor progressively decreases the volume of Initial Sample (deck location 2) that is used to prepare each normalized sample (deck location 6). This reduction in sample volume is then substituted with diluent to maintain the same target volume. The Evaporation Correction Factor represents the relative increase in concentration across an entire plate of 96 samples. For example, if the target concentration is 1.00 mg/mL, and after normalization A1 = 1.00 mg/mL and H12 = 1.05 mg/mL, then the relative increase in concentration across the plate would be 0.05 or 5%. Thus, the recommended Evaporation Correction Factor would be 5%.

How to determine an appropriate Evaporation Correction Factor To determine an appropriate percentage to use for the Evaporation Correction Factor, run a representative normalization protocol to determine the amount of concentration increase observed. The representative normalization run can use an inexpensive reagent, such as bovine serum albumin (BSA). Example 1 describes how to determine the Evaporation Correction Factor from a full plate of normalized results. Example 1. Determining the Evaporation Correction Factor from a full plate of normalization results. Target Concentration = 1.0 mg/mL Normalization Run Results:

Figure Example 1 normalized sample concentration (mg/mL) without an Evaporation Correction Factor

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To calculate the Evaporation Correction Factor for Example 1: 1 Use the following equation to calculate relative inaccuracy (RI) for each of the 96

samples with respect to the target concentration:

where, RIw is the relative inaccuracy for well location w

Cw is the concentration for well location w

Co is the target concentration

2 Plot the relative inaccuracy (RI) values against sample number (n), along with a linear regression line.

3 The equation for the regression line is used to solve for RI when n = 96. This gives the relative increase in concentration across the plate, and thus the recommended Evaporation Correction Factor. The calculated factor for this example is as follows: Evaporation Correction Factor = 0.0007 96-0.0109 = 0.056 = 5.6%

The same process can be followed when using a partial plate of normalized samples, as Example 2 demonstrates. Example 2: Determining the Evaporation Correction Factor from a partial plate of normalization results. Target Concentration = 1.0 mg/mL Normalization Run Results for Partial Plate:

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Figure Example 2 normalized sample concentration (mg/mL) without an Evaporation Correction Factor

To calculate the Evaporation Correction Factor for Example 2: 1 Calculate the relative inaccuracy (RI) for each of the 48 samples with respect to the

target concentration. See Example 1 for the equation to calculate RI. 2 Plot the relative inaccuracy (RI) values against sample number (n), along with a

linear regression line. The following figures show how the sample number (n) would change for this partial plate example.

Figure

Figure Sample run order: sequence in which the samples were normalized

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3 The equation for the regression line is used to solve for RI when n = 96. This gives the relative increase in concentration across the plate, and thus the recommended Evaporation Correction Factor. Evaporation Correction Factor = 0.0007 96-0.0113 = 0.056 = 5.6%

Note: Even though only 48 sample were run in this example, the Evaporation Correction Factor is still calculated by solving the regression equation for n = 96. This is because the Evaporation Correction Factor is expressed as the percent increase across an entire plate of 96 samples. Because we don't have a full plate of results, the correction factor must be determined by extrapolation.

Conditions that can affect evaporation

Three factors that are known to affect the evaporation that is observed when using the Normalization utility are:

1 Atmospheric conditions in the laboratory The temperature and humidity of a laboratory affect the amount of evaporation that is observed with samples that are exposed to atmosphere. Many laboratories have environmental management systems to help maintain constant atmospheric conditions. These environmental controls will help to reduce changes in the amount of evaporation that is observed, but slight changes in the Evaporation Correction Factor may be required to maintain a consistent normalization results, especially with changing seasons.

2 Sample Plate labware type The Initial Sample Plate labware type can have a significant impact on the amount of evaporation that is observed. Different well geometries and volume capacities can impact the rate of evaporation that will be observed. Well geometry. Plates with larger diameter wells provide greater surface area

for interaction with the open atmosphere, leading to faster evaporation rates. Volume capacity. Consider a scenario where Plate 1 has a 100-L capacity and

Plate 2 has a 10-L capacity. If both plates lose 5 L of diluent to evaporation, the resulting concentration in Plate 1 would increase by a factor of ~1.05, while the concentration in Plate 2 would increase by a factor of 2.

3 Solvent The solvent in which the sample is suspended will dramatically affect the evaporation rate. When determining the evaporation rate, use the same sample solvent that will be used during the normalization run using real samples.

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Pipetting accuracy and liquid classes

IMPORTANT Agilent does not guarantee specific accuracy and precision results for the Normalization utility because accuracy and precision are too dependent on the composition and liquid properties of the samples and diluent used.

The following table lists the default liquid classes that the Normalization utility uses.

These default liquid classes should give acceptable accuracy results for most samples types that will be used with the Normalization utility. Pipetting accuracy and precision can be greatly affected by liquid properties, such as viscosity and surface tension. If a sample or diluent has properties that are different from simple aqueous solutions, then these liquid classes might not give results within the expected accuracy and precision range. If unacceptable accuracy and precision results are observed, you may modify these liquid classes to achieve acceptable results. See the About Liquid Classes section of the VWorks Version 4 Automation Control Setup Guide for more information about liquid classes, and instructions on how to modify them to achieve the specific pipetting characteristics.

Liquid class name When to use this liquid class

AM_Normalization_diluent_20-150ul High-volume transfers of diluent (20 to 150 L)

AM_Normalization_diluent_5-20ul Low-volume transfers of diluent (5 to 20 L)

AM_Normalization_sample_20-150ul High-volume transfers of sample (20 to 150 L)

AM_Normalization_sample_5-20ul Low-volume transfers of sample (5 to 20 L)

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Automation movements during the protocol

This section describes the basic automation movements of the AssayMAP Bravo Platform during a Normalization run.

Protocol step Head moves to deck location...

Action

1. Syringe Wash 1 Performs 1 external syringe wash at the wash station.

2. Syringe Drying 1 Performs 4 syringe aspirate-and-dispense cycles above the wash station to cycle air in and out of the syringes. The syringes move over the chimneys after each cycle to remove any droplets that were pushed out of the syringes during the cycle.

3. Initial Tip Transfer

3 Presses on all the 250-L pipette tips from the tip box.

5 Ejects all the pipette tips into the seating station.

4. Single Tip Pickup

5 Uses probe A12 of the head to pick up the next available individual pipette tip.

5. Diluent Transfer

4 Aspirates diluent (well A12) into the pipette tip.

6 Dispenses the diluent into a specific well in the Normalized plate.

6. Sample Transfer

2 Aspirates sample from the Sample plate into the pipette tip.

6 Dispenses the sample into the same well in the Normalized plate that was used for the Diluent Transfer process.

7. Single Tip Eject 3 Ejects the used pipette tip into the tip box. Note: The tip box location matches the well location of the normalized sample that the pipette tip was used to prepare.

8. Additional Transfers

various Repeats steps 2 through 5 for every sample in the Sample plate.

9. Used Tip Pickup

3 Presses on all the used pipette tips from the tip box.

10. Mixing 6 Mixes all the samples in the Normalized plate.

11. Final Tip Ejection

3 Ejects the used pipette tips into the tip box.

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This chapter contains the following topics: Utility description on page 624 Before you start on page 624 Setting up a Reformatting method on page 628 Running the protocol on page 643 Assay development guidelines on page 649

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Utility description

Reformatting v2.0. This utility automatically transfers sample from any well in a 96-well source plate to any well in a 96-well destination plate. You can aspirate repeatedly from the same source and dispense into multiple destination wells. You can draw from multiple source wells and dispense into a single destination well. For example, you can use the utility to perform random hit picking, to pool samples, or to add an internal standard to selected wells in a Serial Dilution plate. You use the Reformatting Method Setup Tool to create a method that specifies the following: List of samples to be reformatted Labware for the source and destination plates Volume to be transferred (51000 L) Liquid-handling parameters, including options to prewet the pipette tips, specify a

blowout volume, choose a liquid class, mix the source and destination wells, and specify a pre-existing volume in the destination wells

The utility uses your saved method to transfer the specified volume of source sample into the designated wells of the destination plate.

Before you start

This topic lists the required hardware, software, and labware for running the Reformatting utility. If you have questions about these items, contact Agilent Customer Service.

Hardware

The AssayMAP Bravo Platform is required for running the AssayMAP protocols. The following figure and table describe the platform components.

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Figure AssayMAP Bravo Platform components

Optional equipment. The following equipment is recommended when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Bravo Risers, 14.6 cm

7 Peltier Thermal Station with STC controller

8 Plate Risers, 2.84 cm (two)

9 Orbital Shaking Station with Control Unit

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Software

The following table lists the minimum software requirements.

For an overview of the software components, see Overview of software architecture on page 15.

Labware

The Reformatting protocol requires the following labware: Full tip box of 96 250-L pipette tips, Agilent 19477-002

Note: A full tip box is required for each run even if the run uses less than 96 pipette tips.

Labware for the source plate and destination plate The following tables provide a complete list of labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Using different pipette tips, such as filtered tips, will result in equipment damage. Ensure that you use the standard 250-L pipette tip (Agilent 19477-002).

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

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Figure Accessory and labware locations on the Bravo deck (top view)

Labware Manufacturer part number*

Deck location options

96 ABgene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 2, 6

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 2, 6

96 Costar 3363, PP Conical Bottom Corning Costar 3363 2, 6

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 2, 6

96 Eppendorf 96-500_V-bottom, Clear PolyPro Eppendorf 96/500 2, 6

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 2, 6

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 2, 6

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 2, 6

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 2, 6

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 2, 6

96 Thermo Matrix 3732, V-bottom, 0.75ml Storage Tubes Thermo Fisher Scientific 3732

2, 6

96 Thermo Matrix 3735, V-bottom, 500l Storage Tubes Thermo Fisher Scientific 3735

2, 6

96 Thermo Matrix 3744, V-bottom, 500l ScrewTop Storage Tubes

Thermo Fisher Scientific 3744

2, 6

96 Waters 186005837, Clear PolyPro Waters 186005837 2, 6

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 2, 6

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 2, 6

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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The unusable volume (also known as dead or overage volume) is the volume that cannot be reliably aspirated from the microplate well due to pipetting limitations that arise from differences in well-bottom geometry. The following table provides some general guidelines on how different microplate geometries can affect this value.

Table Guidelines on how microplate geometry can affect volume requirements

For volume overage recommendations by labware type, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Setting up a Reformatting method

The Method Setup Tool is a Microsoft Excel-based tool that steps you through the process of creating and saving a method file that contains all the information required to run the Reformatting protocol on the AssayMAP Bravo Platform. This tool enables you to define the location of the samples to be transferred, the volumes to be transferred, and the liquid-handling parameters to be used. Note: When you select a method in the Reformatting utility, the form displays the corresponding labware selections and source and destination plate instructions.

Well-bottom geometry

Examples Unusable volume expectations

Flat bottom Greiner 675801 The worst plates for minimizing dead volume. Liquids are attracted to the intersection of the plate bottom and side walls.

Round (U) bottom Greiner 650201, Greiner 650207 Good options for minimizing dead volume. A round bottom forces liquid to collect in the center of the well.

V-bottom and PCR V-Bottom: Greiner 651201, Corning Costar 3363

PCR Plate: Eppendorf 30129300, Greiner 652270

The best plates for minimizing dead volume. Steep side walls force liquid to collect in the center of the well, making it easy to access.

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Figure Reformatting Method Setup Tool

Opening the Method Setup Tool

You can open the tool from the Utility Library or from the Reformatting form.

To open the Method Setup Tool: 1 In the Utility Library, locate the Reformatting banner.

2 Click one of the following buttons: Method Setup Tool. Microsoft Excel starts and displays the Method Setup

Tool. Utility. The Reformatting utility opens.

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In the navigation pane, click Method Setup Tool. Microsoft Excel starts and displays the Method Setup Tool.

IMPORTANT In Microsoft Excel, ensure that you enable content.

Overview of steps in Method Setup Tool

The Method Setup Tool has 4 distinct steps. The following table provides an overview of the steps.

Table Overview of steps in Method Setup Tool

Step Description

1 Select labware Click the green Source plate box, and then select the labware to be placed on the plate riser at deck location 2. Click the green Destination plate box, and then select the labware to be placed on the plate riser at deck location 6 If multiple transfers will be pooled in a single destination well, ensure that the well has sufficient capacity.

2 Specify liquid handling parameters

Enter values for the following inputs: Default value (Range)

Volume (L) to transfer from source to destination Note: <5 L is possible but the accuracy and precision starts to decrease below 5 L.

0 (51000)

Starting volume (L) in source wells 0 (01000)

Pre-existing volume (L) in destination wells 0 (0995)

Source well mix cycles Note: The number of mix cycles is dependent on the volume, viscosity, and size of molecules in the solution being mixed.

0 (0100)

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Step 1) Select labware

For the labware options at each deck location, see Labware on page 626.

To select the labware: 1 In the cell next to Source plate, select the labware that you are placing on the plate

riser at deck location 2 as follows: Click the green cell, and then click the arrow that appears. In the list, click the labware type to be used.

2 In the cell next to Destination plate, select the labware that you are placing on the plate riser at deck location 6 as follows: Click the green cell, and then click the arrow that appears. In the list, click the labware type to be used.

Destination well mix cycles If transferring from multiple source wells into a single destination well, additional off-deck mixing may be required because the mixing volume is based on a single transfer volume.

0 (0100)

Pre-wet tips Yes (No, Yes)

Blowout volume (L) 5 (050)

Liquid class Automatic (presets or custom)

3 Create list of samples to reformat

Use one or a combination of the following options to create and edit the list of samples: Option 1. Select the source and destination well locations for each sample on

the plate maps. Option 2. Load a saved method.

Note: The saved method must be exported in order to use Option 2. Option 3. Manually enter the source and destination locations for each sample

in the sample table.

4 Save method Click Create Method File. The setup tool creates a comma-separated value (.csv) text file that you use to run the utility.

Step Description

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IMPORTANT If transfers from multiple source wells will be pooled in a single destination well, be sure that the destination well has enough capacity to receive these multiple transfers. The software will not alert you to this type of error.

Step 2) Specify liquid handling parameters

Note: Only the cells highlighted in green are editable.

To specify the liquid handling parameters: Type or select the values for the following inputs:

Input Description

Volume (L) to transfer from source to destination

The volume to be aspirated from the specified well in the source plate and then dispensed in the specified well in the destination plate. Note: This value is automatically entered into the list of samples to be reformatted in step 3, option 1 of the Method Setup Tool. You can use step 3, option 3 to edit the volume on a sample-by-sample basis. Default: 0 (L) Range: 51000 (L) Note: Samples <5 L are possible using step 3, option 3, but samples <5 L may not have good accuracy or precision.

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Starting volume (L) in source wells

The initial volume in each well of the source plate before the transfer starts. The software uses this volume for error checking and to calculate the liquid height in the wells to facilitate proper dynamic tip behavior. The software error checking assumes that the volume is the same in each well of the plate. If well volumes vary, enter the smallest volume as the Starting volume. By doing so, the error checking can alert you if you enter a value to be transferred that is larger than the initial volume. Also, you should enter the value for the smallest source well volume so as not to introduce bubbles while mixing. Note that the larger volume wells may not mix as well as the smaller volume wells because the mixing volume is based on the value on the Starting volume in source wells setting. If doing multiple transfers from a single source well, be sure to include enough volume as the error checking assumes only a single transfer. Including a volume overage. Be sure to include sufficient excess volume (dead volume) in the source well to ensure good accuracy and precision. For volume recommendations, see the Labware Reference Guide (workbench Literature Library page). The Starting volume in the source wells is the actual volume in the source well, which should equal the amount to be transferred plus the overage (dead volume). For 96 well plates, the Starting volume in the source wells setting is straightforward. For labware where multiple pipette tips can draw from a common source, the situation is more complex. The Starting volume is still the volume to be transferred plus the overage (dead volume) but the overage is not the overage for the entire common source. Instead, it is the overage for the common source divided by the number of virtual wells in that common source based on a 96 well plate map. For example, a 12-Column Low-Profile Reservoir has 8 virtual wells per

column so the overage used to calculate the Starting volume would be the recommended dead volume per column divided by 8. Similarly, an 8-Row Low-Profile Reservoir has 12 virtual wells per row so the overage used to calculate the Starting volume would be the dead volume per row divided by 12.

For a reformatting method where 50 L is to be transferred per well with a 12-Column Low-Profile Reservoir as the source plate, the starting volume should be: 3 mL / 8 = 375 L + 50 L = 425 L

Default: 0 (L) Range: 01050 (L)

Input Description

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Pre-existing volume (L) in destination wells

The initial volume in each well of the destination plate before the Reformatting utility is run. The software uses this volume for error checking, to calculate mixing volumes, and to calculate the liquid height in the wells to facilitate proper dynamic tip behavior. For 96 well plates, the Pre-existing volume in the destination wells is straightforward but for labware where multiple pipette tips can draw from a common source the situation is more complex. The Pre-existing volume is the total volume in the common source divided by the number of virtual wells in that common source, based on a 96-well plate map. For example, a 12-Column Low-Profile Reservoir has 8 virtual wells per column, so the Pre-existing volume would be the volume in the column divided by 8. Similarly, an 8-Row Low-Profile Reservoir has 12 virtual wells per row, so the Pre-existing volume would be the volume in the row divided by 12. Default: 0 (L) Range: 0995 (L)

Source well mix cycles The number of aspirate-and-dispense cycles used to mix the contents of the wells in the source plate before the sample is aspirated. Mix cycles are not recommended if any of the source wells will be used multiple times, because the mixing calculation assumes the volume in the source well is the Starting volume (L) in source wells value. Therefore, following the initial volume drawn, the mixing volume could be greater than the volume in the well, and the mixing could introduce bubbles. The software assumes that all the wells in the source plate have the same volume. If any well has less volume than the volume set in the Starting volume (L) in source wells field, mixing could introduce bubbles. For additional details, see Automation movements and stepwise guidelines on page 649. Default: 0 Range: 0100

Destination well mix cycles

The number of aspirate-and-dispense cycles used to mix the contents of the wells in the destination plate after the sample is transferred to the destination well. The mixing volume may be too low for destination wells that receive multiple transfers because the mixing volume calculation assumes that the volume in the destination well is a single transfer plus the Pre-existing volume (L) in the destination wells. If your protocol includes multiple source well transfers into a single destination well, Agilent recommends using a shaker to perform additional mixing. For additional details, see Automation movements and stepwise guidelines on page 649. Default: 0 Range: 0100

Input Description

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Pre-wet tips The option to wet the pipette tips with the liquid from the source well before drawing liquid from the source well. Note: Prewetting pipette tips is a common pipetting technique that can increase accuracy and precision in certain situations. For additional details, see Automation movements and stepwise guidelines on page 649. Default: Yes Options: Yes, No

Blowout volume (L) The volume of air to be drawn into the pipette tip before aspirating the sample. After dispensing the transferred sample, the volume of air remaining in the tip is dispensed (blown out) while the tip is still in the well. The blowout is followed by a tip touch on the east and west sides of the well. If the Blowout volume is set to 0, no blowout or tip touches will occur after the transfer is complete. For additional details, see Automation movements and stepwise guidelines on page 649. Default: 5 (L) Range: 050 (L)

Liquid class The pipetting parameters (for example, aspiration and dispense speeds). The liquid class selection strongly influences the pipetting precision and accuracy. This choice only controls the aspiration and dispense of the samples. The mixing speed of the dilutions is fixed at 300 L/sec. Options: (default). Automatically assigns one of the following liquid

classes, based on the volume being transferred. 020 L (AM_250uLTipsLowVol) > 20 L (AM_250uLTipsHighVol) These are good general-purpose liquid classes for most reagents that are used with the AssayMAP system.

Slow Flow (5 L/sec). A slower flow rate is better for viscous solutions. Fast Flow (100 L/sec). A faster flow rate may improve performance for high

organic solutions. If these flow rates do not provide the desired performance, you may enter a custom liquid class. To create a custom liquid class, see To modify or create a custom liquid class: on page 636.

To enter a custom liquid class: In the Liquid class box, type the liquid class name exactly as it appears in the VWorks Liquid Library Editor. Note: The estimated run time (Status box area) may not be accurate for methods that use a custom liquid class.

Input Description

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Pipetting accuracy and liquid classes

IMPORTANT Agilent does not guarantee specific accuracy and precision results for the Reformatting v2.0 utility because accuracy and precision are too dependent on the composition and liquid properties of the solutions used.

Pipetting accuracy and precision can be greatly affected by liquid properties, such as viscosity and surface tension. If unacceptable accuracy and precision results are observed, you may modify these liquid classes to achieve acceptable results. Use the following procedure to modify or create a liquid class.

To modify or create a custom liquid class: 1 In the Reformatting form, click Toggle Full Screen to change the display of the form

so that the VWorks menubar is visible.

Figure VWorks Tools menu and form displayed with full screen off

2 Click Tools > Liquid Library Editor. The Liquid Library Editor opens. If the menubar is not visible in the window, right-click the window and select Menubar in the shortcut menu that appears.

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3 For instructions on how to use the Liquid Library Editor, see the section on specifying pipette speed and accuracy in the VWorks Automation Control Setup Guide.

Figure VWorks Liquid Library Editor window

Step 3) Create list of samples to reformat

You can use any of the following options to create and edit the list of samples: Option 1. Click to add samples Option 2. Load saved method

A saved method must be exported before it is available to load in Option 2. For details on how to export a method, see Exporting and importing AssayMAP methods on page 17.

Option 3. Manual entry You can also use a combination of options. For example, you can enter some sample transfers using option 1 or 2 and then you can add additional sample transfers manually. If you do this, remember to click Update Layouts after you are done. The added samples appear in the Samples to be Reformatted table and in the Source Plate Layout and Destination Plate Layout, as the following figure shows. You can use the manual entry method to modify the volumes in the Samples to be Reformatted table. Note: The Method Setup Tool assigns consecutive sample numbers based on the sequence in which you add the samples.

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If necessary, you can delete samples from the list one at a time using the Delete sample feature. The Method Setup Tool consecutively renumbers the remaining samples in the list based on the sequence in which they were added.

To use the Click to add samples option: 1 Click Start Adding Samples to List.

Note: The button label changes to Stop Adding Samples to List. 2 In the Source Plate Layout, click the well that contains the sample to be moved, and

then click the well in the Destination Plate Layout where this sample will go. As the figure shows, the cells change color in both plate layouts to designate the added samples. The corresponding sample and well designations appear in the Samples to be Reformatted table.

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3 Repeat step 2 to add additional samples to the list. You can draw multiple times from the same source well. See B1 in the

following source plate example. You can dispense multiple times in the same destination well. See C1, H1, and

C2 in the following destination plate example. 4 When you are finished adding samples using Option 1, click Stop Adding Samples

to List. See figure, item 4.

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To use the Load Saved Method option: 1 Make sure you have exported a saved method:

a Use the File > Export Misc File command in the VWorks window. b Ensure that the method file to be exported is a Reformatting v3.0 method.

VWorks Plus default storage location: /VWorks Projects/VWorks/AM Methods/AM Reformatting v3.0

VWorks Standard default storage location: C:\OLSS Projects\VWorks Projects\VWorks\AM Methods\AM Reformatting v3.0

For details, see Exporting and importing AssayMAP methods on page 17. 2 If applicable, click Clear All to refresh the tables in the Method Setup Tool. 3 Click Load Saved Method.

a In the Open dialog box, select the exported method file (*.csv), and then click Open.

b The samples appear in the Source Plate Layout, Destination Plate Layout, and Samples to be Reformatted table.

To use the Manual entry option: 1 Type or copy-and-paste the required information in the Samples to be Reformatted

table. Note: You can edit volumes that you entered using options 1 or 2. If the volume in the Samples to be Reformatted table has been modified so that it does not equal the volume set in Step 2, the corresponding cell turns yellow in the table. The yellow highlight is only a caution for you to verify that these values are intended. If you are copying rows from a table, ensure the content of the cells matches the column order of the Samples to be Reformatted table. See the following figure, item 1.

2 Click Update Layouts to populate the sample information in the Source Plate Layout and Destination Plate Layout (figure, item 2).

3 Notice that the cells change color in both plate layouts to designate the added samples (figure, item 3).

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To delete a sample from the list: 1 Click Delete a Sample from List. See figure, item 1.

The Delete Sample From List dialog box opens. 2 In the Samples to be Reformatted table, click the cell in the Sample Number column

(figure, item 2). Note: Samples can be deleted only one at a time.

3 Click OK in the Delete Sample From List dialog box (figure, item 3). The software updates the Samples to be Reformatted table and the plate layouts. If you delete a sample that is not at the end of the list, the software renumbers the samples remaining in the list in consecutive order.

Step 4) Save method

To save the reformatting method:

1 Click .

2 In the login window, type your VWorks user name and password, and click Log In.

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3 In the Save File dialog box, specify the file name and the storage location, and then click Save.

VWorks Plus default storage location: /VWorks Projects/VWorks/AM Methods/AM Reformatting v3.0

VWorks Standard default storage location: C:\OLSS Projects\VWorks Projects\VWorks\AM Methods\AM Reformatting v3.0

Note: The method file is stored as a record of interest (ROI) in an archive (.csv.roiZip). VWorks cannot load files (.roiZip extension) that have been modified or renamed outside of the Protein Sample Prep Workbench or VWorks software.

4 Click OK when the uploaded successfully message appears. The Method Setup Tool closes.

To display the methods labware selections and preparation instructions do one of the following: Open the Reformatting utility and select the method. For details, see Running the

protocol on page 643. Export the method and view it in Microsoft Excel. For details, see Exporting and

importing AssayMAP methods on page 17.

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Running the protocol

The Reformatting protocol does the following: Washes and dries the syringes. Transfers sample from a 96-well source plate to a 96-well destination plate

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the reformatting setup method. See Setting up a Reformatting method

on page 628. Display the methods labware selections and preparation instructions by doing one

of the following: Open the Reformatting utility and select the method. For details, see Setting

up the protocol on page 644. Export the method and view it in Microsoft Excel. For details, see Exporting

and importing AssayMAP methods on page 17.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Prepare the source and destination plates. Ensure that the type of labware and volumes per well match what is specified in the reformatting method.

If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

If necessary, run the Startup protocol to initialize the AssayMAP Bravo Platform and prime the wash lines. See the System Startup/Shutdown v3.0 User Guide utility.

IMPORTANT To minimize evaporation, fill the labware immediately before run time or keep them covered until you run the protocol.

Setting up the protocol

To set up the protocol: 1 Locate the Reformatting banner in the Utility Library, and then click Utility.

The Reformatting utility opens.

2 If applicable, click Select Experiment ID.

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The Experiments Editor opens.

3 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

4 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

The form displays the method labware selections and preparation instructions. To run the selected method, go to Starting the protocol run on page 645. To create or modify a method, see Setting up a Reformatting method on

page 628. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. You must use pipette tips for the mock run.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

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To start the protocol run: 1 Ensure that the accessories and labware are at the assigned deck locations, as

shown in the Deck Layout image of the form. Make sure the labware are properly seated on the Bravo deck. The Bravo Plate Risers are securely in place at deck locations 2 and 6. The

source plate is seated on the Bravo Plate Riser at deck location 2. The destination plate is seated on the Bravo Plate Riser at deck location 6.

The tip box full of fresh pipette tips is at deck location 3 and the empty 96AM Cartridge & Tip Seating Station is at deck location 5. Note: A full tip box is required for each run even if the run uses less than 96 pipette tips. Note: The protocol will transfer the pipette tips to the seating station.

CAUTION To prevent a potential collision, ensure that no thermal plate insert is on the Peltier Thermal Station installed at deck location 4.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

Note: The run time varies depending on the method. The software updates the run time throughout the course of the run, and the completion time estimate becomes more and more accurate as the run progresses.

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WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

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c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck

match the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

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Assay development guidelines

This topic provides guidelines for adjusting the protocol, and notes about the application. Note: For maximum well capacities and recommended excess volumes in the source plate and destination plate, see the Labware Reference Guide in the Literature Library page of the workbench.

Automation movements and stepwise guidelines

Protocol step

Head moves to deck location

Action

1. Syringe Wash

1 Performs 1 external syringe wash at the wash station.

2. Syringe Drying

1 Performs 4 syringe aspirate-and-dispense cycles above the wash station to cycle air in and out of the syringes. The syringes move over the chimneys after each cycle to remove any droplets that were pushed out of the syringes during the cycle.

3. Initial Tip Transfer

3 Presses on all the 250-L pipette tips from the tip box. Note: The tip box must be full even when using fewer than 96 pipette tips because of the tip-seating force that is used for this step.

5 Ejects the pipette tips into the seating station.

4. Single Tip Pickup

5 Positions the head at an offset from the seating station to use probe A12 to pick up the next available individual pipette tip, starting at H1.

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5. Prep for Transfer

2 Positions the head at an offset to the source plate so that the A12 pipette tip moves to the designated well. If a blowout volume is specified, aspirates an air gap.

This volume is used to ensure that no liquid remains in the pipette tip after the contents of the tip are dispensed. A small blowout volume can help improve precision and accuracy. The magnitude of this effect is usually small for aqueous solutions but can be significant with viscous solutions. Default: 5 L Range: 0-50 L Note: The size of the air gap reduces the possible pipetting volume per pipetting cycle. Larger air gaps have diminishing returns in terms of improved accuracy and precision. The relationship between the size of the air gap and the precision and accuracy is highly dependent on the solution being moved. Agilent recommends starting with the default value to determine if any change is required.

If specified, prewets the A12 pipette tip in the designated source well. Prewetting pipette tips is best practice for good pipetting accuracy and precision. Default: Yes Options: Yes, No

If specified, mixes the sample in the source well the specified number of cycles. The volume used for the mixing process is automatically calculated using the following formula. Mixing formula: If (x - 50 L) > Tip Capacity, then Mixing Volume = Tip Capacity If 50 L x Syringe/Tip Capacity,

then Mixing Volume = 0.75x If x < 50 L,

then Mixing Volume = 0.5x where, x is the final volume in the destination wells, and maximum 250 L Tip Capacity = 140 L

Protocol step

Head moves to deck location

Action

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Note: The mix cycle default is set at 0 to ensure that mixing is not done

unintentionally. If samples are centrifuged before reformatting, mixing would not be wanted.

Generally, aqueous solutions are well mixed by 5 cycles if the volume is <200 L. More viscus and larger volume solutions require more mix cycles, which must be determined empirically.

The more mix cycles you use, the longer the run time.

IMPORTANT Mix cycles are not recommended if any of the source wells will be used multiple times, as the mixing calculation assumes the volume in the source well is the Starting volume (L) in source wells value. Therefore, after aspirating the initial volume, the mixing volume could be greater than the volume remaining in the well, and the mixing could introduce bubbles.

IMPORTANT The software assumes that all the wells in the source plate have the same volume. If any well has less volume than the volume set in the Starting volume (L) in source wells field, mixing could introduce bubbles.

6. Transfer 2 Aspirates the specified volume from the source plate well into the A12 pipette tip.

Protocol step

Head moves to deck location

Action

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6 Positions the head at an offset to the destination plate so that the A12 pipette tip moves to the designated well. Dispenses the sample into the destination well. If specified, mixes the sample in the well the specified number of cycles.

The volume used for the mixing process is automatically calculated using the following formula. Mixing formula: If (x - 50 L) > Tip Capacity, then Mixing Volume = Tip Capacity If 50 L x Syringe/Tip Capacity,

then Mixing Volume = 0.75x If x < 50 L,

then Mixing Volume = 0.5x where, x is the final volume in the destination wells, and maximum 250 L Tip Capacity = 140 L Note: The mix cycle default is set at 0 to ensure that mixing is not done

unintentionally, and is typically only done if there is a pre-existing volume in the destination well.

Generally, aqueous solutions are well mixed by 5 cycles if the volume is <200 L. More viscus and larger volume solutions require more mix cycles, which must be determined empirically.

The more mix cycles you use, the longer the run time. The mixing volume may be far too low for destination wells that receive multiple transfers because the mixing volume calculation assumes that the volume in the destination well is the single transfer volume plus the pre- existing volume in the destination well.

If a blowout volume is specified, the head raises to near the top of the well, and the specified volume of air is dispensed followed by a tip touch on the east and west sides of the well.

7. Single Tip Eject

3 Ejects the used pipette tip into the tip box

8. Additional Transfers

multiple Repeats processes 3 through 6 for every designated well in the source plate.

9. Park Head 1 Moves to a safe height above the wash station.

Protocol step

Head moves to deck location

Action

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Utility description

Serial Dilution v3.0. This utility enables you to automatically generate serial dilutions in a 96-well microplate. You create a method using the Serial Dilution Method Setup Tool. The utility uses your method to transfer the appropriate volume of diluent and source sample into the designated wells of the serial dilution plate to create the desired dilution series. The serial dilution method specifies: Whether the dilution series progresses by columns or rows and the direction of the

series (such as, top to bottom, bottom to top, left to right, or right to left) The number of steps in the dilution series and the number of dilution replicates

(2 to 24 dilutions and 1 to 5 replicates) The final volume of the serial dilution (5500 L) If the utility will change pipette tips after every dilution step, or use the same

pipette tips throughout the dilution series Liquid-handling parameters, including the number of mix cycles after every

dilution, whether to prewet the pipette tips, the blowout volume, and the choice of liquid class

If a constant volume (for example, internal standard) will be added to the dilution series after running the utility

Before you start

This topic lists the required hardware, software, labware, and solutions for running the Serial Dilution protocol. If you have questions about these items, contact Agilent Customer Service.

Hardware

The AssayMAP Bravo Platform is required for running the AssayMAP protocols. The following figure and table describe the platform components.

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Figure AssayMAP Bravo Platform components

Optional equipment. The following equipment is recommended when preparing the samples and reagents: Microplate centrifuge, such as the Agilent Microplate Centrifuge or equivalent Microplate sealer, such as the Agilent PlateLoc Thermal Microplate Sealer or

equivalent

Software

The following table lists the minimum software requirements.

Item Required hardware

1 Gripper upgrade

2 Bravo 96AM Head

3 96AM Wash Station

4 Pump Module 2.0 and two carboys

5 96AM Cartridge & Tip Seating Station

6 Bravo Risers, 14.6 cm

7 Peltier Thermal Station with STC controller

8 Plate Risers, 2.84 cm (two)

9 Orbital Shaking Station with Control Unit

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For an overview of the software components, see Overview of software architecture on page 15.

Labware

The Serial Dilution protocol requires the following labware: Full tip box of 96 250-L pipette tips, Agilent 19477-002 Labware for the diluent reservoir and serial dilution plate The following table provides a complete list of the labware options and the corresponding deck locations. The following figure shows the nine Bravo deck locations for labware.

CAUTION Using different pipette tips, such as filtered tips, will result in equipment damage. Ensure that you use the standard 250-L pipette tip (Agilent 19477-002).

CAUTION Use only the labware specified for each deck location. Using different labware or placing labware at unapproved deck locations can cause a collision resulting in equipment damage.

Figure Accessory and labware locations on the Bravo deck (top view)

Software Version

Agilent VWorks Plus (compliance-enabled edition) or VWorks Standard

14.1.1

Agilent Protein Sample Prep Workbench 4.0

Microsoft Excel Required for the reagent volume calculators and method setup tools.

Microsoft Office 365 32-bit edition

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The unusable volume (also known as dead or overage volume) is the volume that cannot be reliably aspirated from the microplate well due to pipetting limitations that arise from differences in well-bottom geometry. The following table provides some general guidelines on how different microplate geometries can affect this value.

Labware Manufacturer part number*

Deck location options

8 Row, Low Profile Reservoir, Natural PP Agilent 201282-100 2

12 Column, Low Profile Reservoir, Natural PP Agilent 201280-100 2

96 V11 Manual Fill Reservoir Agilent G5498B#049 2

Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom

Agilent 201254-100 2

96 AbGene 1127, 1mL Deep Well, Square Well, Round Bottom

ABgene AB-1127 2, 6

96 Costar 3363, PP Conical Bottom Corning Costar 3363 2, 6

96 Eppendorf 30129300, PCR, Full Skirt, PolyPro Eppendorf 30129300 2, 6

96 Eppendorf 96-500_V-bottom, Clear PolyPro Eppendorf 96/500 2, 6

96 Bio-Rad PCR, Hard-Shell, Low-Profile, Full Skirt Bio-Rad HSP-9611 2, 6

96 Greiner 650201_U-Bottom, Clear PolyPro Greiner 650201 2, 6

96 Greiner 650207_U-Bottom, White PolyPro Greiner 650207 2, 6

96 Greiner 651201_V-Bottom, Clear PolyPro Greiner 651201 2, 6

96 Greiner 652270, PCR, Full Skirt, PolyPro Greiner 652270 2, 6

96 Greiner 675801, Half Area, Flat-Bottom, UV Star Greiner 675801 6

96 Thermo Matrix 3732, V-bottom, 0.75ml Storage Tubes Thermo Fisher Scientific 3732

2, 6

96 Thermo Matrix 3735, V-bottom, 500l Storage Tubes Thermo Fisher Scientific 3735

2, 6

96 Thermo Matrix 3744, V-bottom, 500l ScrewTop Storage Tubes

Thermo Fisher Scientific 3744

2, 6

96 Waters 186005837, Clear PolyPro Waters 186005837 2, 6

*For dimensionally equivalent alternatives and other details about the labware, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

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Table Guidelines on how microplate geometry can affect volume requirements

For volume overage recommendations by labware type, see the Labware Reference Guide in the Literature Library page of the Protein Sample Prep Workbench.

Solutions

The Serial Dilution utility requires the following solutions: Diluent to adjust the sample concentration Purified water for the wash station

Setting up a Serial Dilution method

The Method Setup Tool is a Microsoft Excel-based tool that steps you through the process of creating and saving a method file that contains all the information required to run the Serial Dilution protocol on the AssayMAP Bravo Platform. This tool uses formulas to calculate volume requirements for the samples and diluent based on your input. Note: When you select a method in the Serial Dilution utility, the form displays the corresponding labware selections and diluent preparation instructions.

Well-bottom geometry

Examples Unusable volume expectations

Flat bottom Greiner 675801 Generally, the worst plates for minimizing dead volume. Liquids are attracted to the intersection of the plate bottom and side walls.

Round (U) bottom Greiner 650201, Greiner 650207 Generally, good options for minimizing dead volume. A round bottom forces liquid to collect in the center of the well.

V-bottom and PCR V-Bottom: Greiner 651201, Corning Costar 3363

PCR Plate: Eppendorf 30129300, Greiner 652270

Generally, the best plates for minimizing dead volume. Steep side walls force liquid to collect in the center of the well, making it easy to access.

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Figure Serial Dilution Method Setup Tool

Opening the Method Setup Tool

You can open the tool from the Utility Library or from the Serial Dilution form.

To open the Method Setup Tool: 1 In the Utility Library, locate the Serial Dilution banner.

2 Click one of the following buttons: Method Setup Tool. Microsoft Excel starts and displays the Method Setup

Tool. Utility. The Serial Dilution utility opens.

In the navigation pane, click Method Setup Tool. Microsoft Excel starts and displays the Method Setup Tool.

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IMPORTANT In Microsoft Excel, ensure that you enable content.

Overview of steps in Method Setup Tool

The Method Setup Tool has 6 distinct steps. The following table provides an overview of the steps.

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Table Overview of steps in Method Setup Tool

Step Description

1 Set dilution parameters

Enter values for the following inputs: Default value (Range)

Final concentration of dilution 1 0 (>0)

Optional volume (L) to be added 0 (0 to < Final volume of dilution series)

Final volume (L) of dilution series 0 (5500)

Direction of dilution top to bottom (top to bottom, bottom to top, left to right, right to left)

Number of replicates 1 (15)

Mix cycles after each dilution 10 (0100)

Change pipette tips after each dilution Yes (No/Yes)

Pre-wet pipette tips before transfer Yes (No/Yes)

Blowout volume (L) 5 (050)

Liquid class Automatic (preset or custom)

Serial dilution plate Labware on page 656

Diluent reservoir Labware on page 656

2 Define dilution concentrations

Enter the final target concentration for each serial dilution step.

3 Select the starting well of the dilution series

Click Select Starting Well, and then click the cell in the Serial Dilution Plate map where the replicate 1 of dilution 1 will be located. The setup tool will display the plate layout based on the input provided in steps 1 and 2.

4 Calculate the sample and diluent volumes

Click Calculate Volumes. The setup tool calculates the required volumes of sample and diluent based on the input provided in steps 1 to 3.

5 Correct these errors

Resolve any errors that the setup tool highlights.

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IMPORTANT If you change anything in step 1 or 2 after completing step 3 or 4, make sure that you click Clear Steps 3 and 4 Only and redo steps 3 and 4.

Step 1) Set dilution parameters

Note: Only the cells highlighted in green are editable.

To set the dilution parameters: 1 To clear any data from the Serial Dilution Method Setup Tool, click Clear All (upper

right corner). 2 Specify the following values in the green cells:

For additional details on these settings, see Assay development guidelines on page 679.

6 Create the method file

Click Create Serial Dilution Method. The setup tool creates a comma-separated value (.csv) text file that you use to run the utility. After you save the method, the Method Setup Tool closes. When you select the method in the Serial Dilution utility, the form displays the corresponding labware selections and diluent preparation instructions. Follow the instructions that appear in the form to prepare the serial dilution and diluent plates.

Step Description

Input Description

Final concentration of dilution 1

Concentration units In the Input column, verify that the unit of measure for the sample concentration is correct. If necessary, type the correct units (default is mg/mL). This input is used to populate other sections of the form. This value has no impact on the subsequent data or volume calculations.

Concentration value In the Value column, type the final concentration of the most concentrated level in the serial dilution series (e.g., Dilution 1 in Step 2).

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Optional volume (L) to be added

The volume to be added manually or using the Reagent Transfer utility after the Serial Dilution run, for example, an internal standard. The set up tool uses this value to reduce the volume of the serial dilution to the final volume minus the optional volume to be added. The set up tool also uses this value to increase the concentration of each serial dilution step (see Post Run cells in Step 2) so that when the optional volume is added, the final serial dilution volumes and concentrations match the desired final serial dilution series volumes and concentrations.

IMPORTANT The value you enter here must be less than the Final volume. The recommended minimum volume is 5 L.

For example, if the Final volume = 50 L and Optional volume to add later = 10 L, the Serial Dilution steps will have a volume of 40 L after the run, at which point the optional 10 L will be added for a total volume of 50 L. Default: 0 (L) Range: 050 (L)

Final volume (L) of dilution series

The final volume of each step in the dilution series. This includes the Optional volume (L) to be added. Note: The practical range is approximately 20500 L, depending on the number and concentration of the dilution steps. The high end of the range is limited by well capacities and the high volume transfers required during the early steps of the dilution series compared to the final volume of the dilution steps. The low end of the range is limited by the requirement that the transfers be 5 L or greater to ensure high precision and accuracy. Large-volume dilution series require more mix cycles (up to 100 for the largest volume serial dilutions) because of the limited volume that can be used for each mix cycle. The results for large volume dilution series may not be as accurate as smaller volume dilution series. Default: 0 (L) Range: 5500 (L)

Direction of dilution The direction in which the dilution series should progress. The dilution series follows a serpentine pattern. See the example in Step 3) Select the starting well of the dilution series on page 668. Default: top to bottom Options: top to bottom, bottom to top, left to right, right to left

Input Description

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Number of replicates The number of replicates of each dilution step in the dilution series. Note: This cell turns yellow if you select 5 replicates. The yellow cautions you that the serial dilution may not fit on the serial dilution plate, depending on the number of steps in the dilution series. The maximum number of dilution steps with 5 replicates is as follows: 16 for top-to-bottom or bottom-to-top dilution series 12 for left-to-right or right-to-left dilution series The Number of replicates value determines the total number of wells that will be prepared for each step in the dilution series. For example, if the number of replicates is set to 2, then on run completion, there will be two wells containing each concentration in the series. Default: 1 Range: 15

Mix cycles after each dilution

The number of aspirate-and-dispense cycles used to mix the contents of the wells in the serial dilution plate. Default: 10 Range: 0100

Change pipette tips after each dilution

The option to change pipette tips before transferring the sample for each dilution step. Default: Yes Options: Yes, No

Pre-wet pipette tips before transfer

The option to wet the pipette tips with the sample before aspirating the sample. Note: Prewetting the pipette tips is a common pipetting technique that can increase accuracy in certain situations. Default: Yes Options: Yes, No

Blowout volume (L) The volume of air to be drawn into the pipette tips before aspirating the sample. After dispensing the transferred sample, the volume of air remaining in the pipette tips is dispensed (blown out) while the tips are still in the wells. The blowout is followed by a tip touch on the east and west sides of the well. If the Blowout volume is set to 0, no blowout or tip touches will occur. Default: 5 (L) Range: 050 (L)

Input Description

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Liquid class The pipetting parameters (for example, aspiration and dispense speed). The liquid class selection strongly influences the pipetting precision and accuracy. This choice only controls the aspiration and dispense of the samples and diluent. The mixing speed of the dilutions is fixed at 300 L/sec. Options: (default). Automatically assigns one of the following liquid

classes, based on the volume being transferred. 020 L (AM_250uLTipsLowVol) > 20 L (AM_250uLTipsHighVol) These are good general-purpose liquid classes for most reagents that are used with the AssayMAP system.

Slow Flow (5 L/sec). A slower flow rate is better for viscous solutions. Fast Flow (100 L/sec). A faster flow rate may improve performance for high

organic solutions. If these flow rates do not provide the desired performance, you may enter a custom liquid class. To create a custom liquid class, you use the VWorks Liquid Library Editor. For details on how to open the VWorks Liquid Library Editor, see To create a custom liquid class: on page 666.

To enter a custom liquid class: In the Liquid Class box, type the liquid class name exactly as it appears in the VWorks Liquid Library Editor. Note: The estimated run time (Status box area) may not be accurate for methods that use a custom liquid class.

Serial dilution plate The labware that you are placing on the plate riser at deck location 6. For options, see Labware on page 656. For the capacity limits and the volume overage recommended for each labware, see the Labware Reference Guide in the Literature Library page of the workbench. Note: The intermediate volumes required to generate the serial dilution will be larger than the final volume of the serial dilution. If you have chosen a plate type whose capacity is lower than required, you will be alerted in steps 4 and 5.

Diluent reservoir The labware that you are placing on the plate riser at deck location 2. For options, see Labware on page 656. For the capacity limits and the volume overage recommended for each labware, see the Labware Reference Guide in the Literature Library page of the workbench. Be sure to choose a diluent reservoir that can hold the volume required for the serial dilution. The volume of diluent and the location where it should be placed will be given in step 7 of the Method Setup Tool. The plate type chosen must have enough well capacity to hold the dilution indicated in step 7 plus the required well overage. For volume guidelines, see Preparing the diluent and samples on page 672. Note: If the selected labware cannot hold the volume of diluent required for the serial dilution, an error message will appear in steps 4 and 5.

Input Description

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Pipetting accuracy and liquid classes

IMPORTANT Agilent Technologies does not guarantee specific accuracy and precision results for the Serial Dilution v2.0 utility because accuracy and precision are too dependent on the composition and liquid properties of the solutions used.

Pipetting accuracy and precision can be greatly affected by liquid properties, such as viscosity and surface tension. If unacceptable accuracy and precision results are observed, you may modify these liquid classes to achieve acceptable results. Use the following procedure to modify or create a liquid class.

To create a custom liquid class: 1 In the Serial Dilutionform, click Toggle Full Screento change the display of the form

so that the VWorks menubar and toolbar are visible, as the following figure shows.

Figure VWorks Tools menu and form displayed with full screen off

2 Click Tools > Liquid Library Editor. The Liquid Library Editor opens. If the menubar is not visible in the window, right-click the window and select Menubar in the shortcut menu that appears.

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3 For instructions on how to use the Liquid Library Editor, see the section on specifying pipette speed and accuracy in the VWorks Automation Control Setup Guide.

Figure VWorks Liquid Library Editor window

Step 2) Define dilution concentrations

To define the concentration of the serial dilution steps: In the green boxes in the Final column, enter the desired concentration of each serial dilution step. The method setup tool automatically enters the dilution concentration for dilution 1 when you enter the Final concentration of dilution 1 (Step 1) Set dilution parameters on page 662). You enter all the other concentrations in the dilution series directly in each cell or by entering a formula in the cell. You can use the Excel fill series function with formulas to quickly generate a serial dilution. You can generate serial dilutions with 2 to 24 steps. You can also generate blanks by putting zero as a dilution concentration. Note: If you enter zero as a concentration, an error message appears and says Negative or zero values cannot be plotted correctly on log charts. Only positive values can be interpreted on a logarithmic scale. To continue and use the zero, click OK. The graph will not include the zero volume but the method will work. The Post Run column contains non-editable cells that show the concentration of the serial dilution series after the run is complete.

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If step 1 Optional volume to be added is >0, the Post Run column will show the concentration of the serial dilution steps after the serial dilution utility is run but before the optional volume is added. The concentrations entered in the Final column represent the concentration of the serial dilution after adding the optional volume.

If step 1 Optional volume to be added is 0, the concentrations entered in the Final column will be the same as the those in the Post Run column.

As you define the concentrations, the graph displays the corresponding log-log plot of the dilution series concentrations. You can use the graph to verify that your dilution formula will yield the expected results. The graph can help you visualize the distribution curve and identify gaps between dilution steps.

Step 3) Select the starting well of the dilution series

IMPORTANT If you change anything in step 1 or 2 after starting step 3 or 4, make sure that you click Clear Steps 3 and 4 Only and redo steps 3 and 4.

To define the layout of the serial dilution plate: 1 Click Select Starting Well (figure, item 1) and then click the corresponding cell in the

Serial Dilution Plate map where the replicate 1 of dilution 1 will be located (figure, item 2).

2 When the Choose Starting Location dialog box opens, displaying a formula, click OK (figure, item 3).

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The layout for the dilution series appears in the Serial Dilution Plate map, displaying the selected number of replicates in the specified layout (row or column).

Figure Example of a left-to-right layout by row for a dilution series with 4 replicates

Figure Example of a top-to-bottom layout by column for a dilution series with 4 replicates

Step 4) Calculate the sample and diluent volumes

To calculate the sample and diluent volumes: Click Calculate Volumes. The tool uses the information provided in the previous steps to calculate the sample and diluent volumes that will be transferred into each well to achieve the specified serial dilution series. The calculated volumes (L) appear in the following areas: Sample Volume plate map Diluent Volume plate map

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Step 5) Correct these errors

Although you may enter values that are not possible or practical given the selected labware or specified concentrations and volumes, the setup tool will flag such values as errors. You must resolve any errors before you can proceed to the next step in the setup tool.

IMPORTANT If you change anything in step 1 or 2 at this point, click Clear Steps 3 and 4 Only and redo steps 3 and 4.

Step 6) Create the method file

To create the Serial Dilution method file: 1 Click Create Serial Dilution Method. 2 In the login window, type your VWorks user name and password, and click Log In.

3 In the Save File dialog box, specify the file name and the storage location, and then click Save.

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VWorks Plus default storage location: /VWorks Projects/VWorks/AM Methods/AM Serial Dilution v3.0

VWorks Standard default storage location: C:\OLSS Projects\VWorks Projects\VWorks\AM Methods\AM Serial Dilution v3.0

Note: The method file is stored as a record of interest (ROI) in an archive (.csv.roiZip). VWorks cannot load files (.roiZip extension) that have been modified or renamed outside of the Protein Sample Prep Workbench or VWorks software.

4 Click OK when the uploaded successfully message appears. The Method Setup Tool closes.

Next steps: If you want to view the method in Microsoft Excel, see Exporting and importing AssayMAP methods on page 17 If you are ready to prepare the serial dilution plates, see Preparing the diluent and samples on page 672.

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Preparing the diluent and samples

Before you start

If you have not already done so, display the methods labware selections and diluent and sample preparation instructions by doing one of the following: Open the Serial Dilution utility and select the method. For details, see Setting up

the protocol on page 675. Export the method and view it in Microsoft Excel. For details, see Exporting and

importing AssayMAP methods on page 17.

CAUTION Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the selections in the method exactly match the physical labware present on the Bravo deck.

Preparing the diluent reservoir and serial dilution plates

To prepare the diluent reservoir plate: Follow the methods instructions to prepare the diluent volume (L) necessary for each dilution series replicate and the well locations where this diluent volume should be placed. In addition to the diluent volume required to generate the serial dilution curve, some volume overage is required to account for the dead volume in the plate well. For recommended overage volumes and well capacities, see the Labware Reference Guide (workbench Literature Library page). For 96-well plates, this volume calculation is straightforward in that it is the

volume required plus the recommended overage for the well. For plates where multiple wells on a 96-well plate map are combined in a common

well, the situation can be more complex. If only a single serial-dilution series will draw from the common well (e.g.,

using an 8-channel reservoir where the serial dilutions go from left to right) the situation is the same as in a 96-well plate where the volume required in the common well is the volume indicated in step 7 plus the required overage for the common well.

If more than 1 serial dilution will draw from a common well (e.g., using an 8-channel reservoir where the serial dilutions are going from top to bottom and you have more than one replicate) you would add all the required volumes indicated in step 7 plus the required overage for the common well.

If necessary, use the Reagent Transfer utility to transfer the diluent into the appropriate labware type. For instructions, see Reagent Transfer v3.0 User Guide on page 525.

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To prepare the serial dilution plate: Follow the methods instructions to prepare the volume (L) and concentration of the first dilution for each dilution series replicate and the well locations where this starting volume should be placed. Ensure that the samples in the serial dilution plate match the volume and well positions specified in the method created using the Method Setup Tool. If necessary, use the Reformatting utility to transfer the samples into the appropriate labware type. For instructions, see the Reformatting v3.0 User Guide.

IMPORTANT To minimize evaporation, fill the labware immediately before run time or keep them covered until you run the protocol.

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Running the protocol

The Serial Dilution protocol does the following: Washes and dries the syringes. Transfers the appropriate volume of diluent and source sample into the

designated wells of the serial dilution plate.

Experiment ID and method requirements

Each workbench application and utility has an Experiment Settings section that allows you to select an experiment ID and a method. An experiment ID is a database record that captures the steps executed and the

settings used during each run of an application or utility. Any errors that may have occurred during a run are also recorded. To create an experiment ID, you open the Experiments Editor by clicking

in any Workbench app or utility. For details, go to the Literature Library and open Using the Protein Sample Prep Workbench. In the browser that opens, click Using Experiment IDs.

A method is a comprehensive collection of saved settings for an application or utility, which you can use to run the application or utility.

Experiment IDs and methods are required for compliance-enabled VWorks editions and optional for noncompliance-enabled VWorks editions.

Before you start

Ensure that you: Prepare the serial dilution setup method. See Setting up a Serial Dilution method

on page 658. Display the methods labware selections and diluent preparation instructions by

doing one of the following: Open the Serial Dilution utility and select the method. For details, see Setting

up the protocol on page 675. Export the method and view it in Microsoft Excel. For details, see Exporting

and importing AssayMAP methods on page 17.

VWorks edition Experiment ID and method selection

VWorks Plus Required

VWorks Standard Optional

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Prepare the diluent reservoir and serial dilution plates. See Preparing the diluent and samples on page 672.

If applicable, make sure that you know which experiment ID to use to record the steps executed during the utility and app runs.

If necessary, run the Startup protocol to initialize the AssayMAP Bravo Platform and prime the wash lines. See the System Startup/Shutdown v3.0 User Guide on page 574.

Setting up the protocol

To set up the protocol: 1 Locate the Serial Dilution banner in the Utility Library, and then click Utility.

The Serial Dilution utility opens.

2 If applicable, click Select Experiment ID.

The Experiments Editor opens.

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3 Select the Experiment ID that you want to use to record the steps performed during this application run, and then click Use Selected. The Experiments Editor closes.

4 In the form, click Select Method to locate and select a method. In the Open File dialog box, select the method, and click Open.

The form displays the method labware selections and preparation instructions. To run the selected method, go to Starting the protocol run on page 676. To create or modify a method, see Setting up a Serial Dilution method on

page 658. VWorks Plus. Administrator or technician privileges are required to create and modify methods.

About performing a mock run (optional)

If you are unfamiliar with the protocol and would like to see how it operates before running it with valuable samples and reagents, you can perform a mock run. A mock run uses empty or water-filled labware and source bottles. You prepare for a mock run the same way you would prepare for a real protocol run, except that you use empty labware for a totally dry run or labware containing water for a wet run. You must use pipette tips for the mock run.

Starting the protocol run

WARNING The probes of the Bravo 96AM Head are sharp and can scratch you if they brush across your hand. A probe scratch can expose you to any contaminants remaining on the probes. Be careful to avoid touching the probes.

To start the protocol run: 1 Ensure that the accessories are at the assigned deck locations, as shown in the

Deck Layout image of the form.

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The Bravo Plate Risers are securely in place at deck locations 2 and 6. The diluent reservoir plate is seated on the Bravo Plate Riser at deck location 2. The serial dilution plate is seated on the Bravo Plate Riser at deck location 6.

The tip box full of fresh pipette tips is at deck location 3 and the empty 96AM Cartridge & Tip Seating Station is at deck location 5. Note: A full tip box is required for each run even if the run uses less than 96 pipette tips. Note: The protocol will transfer the pipette tips to the seating station.

CAUTION To prevent a potential collision, ensure that no thermal plate insert is on the Peltier Thermal Station installed at deck location 4.

CAUTION Incorrect labware selections and improperly seated labware can cause hardware collisions, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the Bravo deck. Also ensure that all labware are properly seated within the alignment features of their respective platepads.

2 Click to start the run.

To monitor the progress of the run, check the Status box.

Note: The run time varies depending on the method. The software updates the run time throughout the course of the run, and the completion time estimate becomes more and more accurate as the run progresses.

After the Serial Dilution run. You can run the Reagent Transfer utility to add the Optional volume to be added to the serial dilution plate, if specified in your method.

WARNING To stop a run in an emergency, use the hardware Emergency Stop button.

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To pause the run, click Pause. The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens. For details, see Emergency stops and pauses on page 683. To troubleshoot errors, see the Error Recovery Guide and the Bravo Platform User Guide in the Literature Library page of the Protein Sample Prep Workbench.

Adding an experiment ID note after the run

After the protocol run ends or during a pause, you can add a note to the experiment ID. For example, a note can describe any observations during the run or any offline steps that are being executed. The notes that you add will appear in any reports generated for the experiment ID.

To add a note to an open experiment ID: 1 While the experiment ID is still selected in the Experiment Settings area, click

. The Add Note dialog box opens.

2 In the Note area, type the note, and then click OK.

For detailed instructions on working with Experiment IDs, see Using Experiment IDs on page 23.

Cleaning up

Note: Pipette tips that remain in the seating station after the run completion are unused, and they can be reclaimed for future use.

To clean up after a run: 1 Remove used labware from the deck. 2 Discard leftover reagents appropriately. 3 Optional. Conduct stringent washing of the syringes:

a Open the Syringe Wash utility .

b If applicable, click Select Experiment ID to open the Experiments Editor.

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c In the Experiments Editor, select the Experiment ID that you want to use to capture the steps performed during this utility run, and then click Use Selected.

d Click Select Method to select and load the method for this utility. e Confirm that the labware and accessories on the AssayMAP Bravo deck

match the display in the Deck Layout area of the form.

f Click to start the run.

WARNING Make sure you discard the chemical waste and used labware according to your labs waste disposal procedures and in compliance with all local, state, and federal safety regulations.

To shut down at the end of the day: Run the System Shutdown utility. See System Startup/Shutdown v3.0 User Guide on page 574.

Assay development guidelines

This topic provides guidelines for adjusting the protocol, and notes about the application. Note: For maximum well capacities and overage recommendations in the serial dilution plates, see the Labware Reference Guide in the Literature Library of the Protein Sample Prep Workbench.

Automation movements and stepwise guidelines

This section describes the basic automation movements of the AssayMAP Bravo Platform during the Serial Dilution run.

Protocol step

Head moves to deck location

Action

1. Syringe Wash

1 Performs 1 external syringe wash at the wash station.

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2. Syringe Drying

1 Performs 4 syringe aspirate-and-dispense cycles above the wash station to cycle air in and out of the syringes. The syringes move over the chimneys after each cycle to remove any droplets that were pushed out of the syringes during the cycle.

3. Initial Tip Transfer

3 Presses on all the 250-L pipette tips from the tip box. Note: The tip box must be full even when using fewer than 96 pipette tips because the tip-seating force has been set assuming that a full rack of pipette tips are being seated.

5 Ejects the pipette tips into the seating station.

4. Offset Head Tip Pickup

5 Positions the head at an offset from the seating station to pick up between 1 to 5 pipette tips, depending on the number of replicates, from the next available column or row of pipette tips. Picks up tips in row format for top-to-bottom or bottom-to-top dilution series. Picks up tips in column format for left-to-right or right-to-left dilution.

5. Diluent Transfer

2 Moves to the diluent reservoir. If a blowout volume is specified, aspirates an air gap.

This volume is used to ensure that no liquid remains in the pipette tip after the contents of the tip are dispensed. A small blowout volume can help improve precision and accuracy. The magnitude of this effect is usually small for aqueous solutions but can be significant with viscous solutions. Default: 5 L Range: 050 L Note: The size of the air gap reduces the possible pipetting volume per pipetting cycle. Larger air gaps have diminishing returns in terms of improved accuracy and precision. The relationship between the size of the air gap and the precision and accuracy is highly dependent on the solution being moved. Agilent recommends starting with the default value to determine if any change is required.

If specified, prewets the pipette tips in the designated source wells. Prewetting pipette tips is best practice for good pipetting accuracy and precision. Default: Yes Options: Yes, No

Aspirates diluent into the pipette tips.

6 Dispenses the diluent into the designated wells in the serial dilution plate. If a blowout volume is specified, the head raises to near the top of the well,

and the specified volume of air is dispensed followed by a tip touch on the east and west sides of the well.

This aspirate-and-dispense process repeats using the same pipette tips until all the wells in the serial dilution plate have the specified amount of diluent. If a blowout is specified, an air gap is aspirated at the start of each aspirate task and a blowout and tip touch occur after each dispense task.

Protocol step

Head moves to deck location

Action

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18 Serial Dilution v3.0 User Guide Assay development guidelines

AssayMAP Protein Sample Prep Workbench User Guide

6. Change Tips

3 Ejects the used pipette tips into the tip box.

5 Picks up between 1 to 5 pipette tips, depending on the number of replicates, from the next available column or row of pipette tips in the seating station. Picks up tips in row format for top-to-bottom or bottom-to-top dilution series. Picks up tips in column format for left-to-right or right-to-left dilution.

7. Serial Dilution Transfer

6 Moves to the lowest concentration dilution in the serial dilution plate. If a blowout volume is specified, aspirates an air gap. For details, see protocol

step 4, above. If specified, prewets the pipette tips in the designated wells. Aspirates the sample into the pipette tips, and then dispenses the sample into

the next set of dilution wells in the serial dilution plate. If specified, mixes the well contents for the set number of cycles.

The volume used for the mixing process is automatically calculated using the following formula. If (x - 50 L) > Tip Capacity, then Mixing Volume = Tip Capacity If 50 L x Syringe/Tip Capacity,

then Mixing Volume = 0.75x If x < 50 L,

then Mixing Volume = 0.5x where, x is the final volume in the destination wells, and maximum 250 L Tip Capacity = 140 L Default: 10 Range: 0100 Note: Generally, aqueous solutions are well mixed by 10 cycles if the maximum

intermediate volume in the serial dilution (see step 4 in the setup tool) is > 200 L. The intermediate serial dilution volumes are always higher than the final volume. More viscus and larger volume solutions require more mix cycles, which must be determined empirically.

The more mix cycles you use, the longer the run time. If a blowout volume is specified, the head raises to near the top of the well,

and the specified volume of air is dispensed followed by a tip touch on the east and west sides of the well.

8. Optional Change Tips

3 If the change tips option is specified, ejects the used pipette tips into the tip box. If the change tips option is not specified, this step is skipped and the same set of pipette tips are used for all the serial dilution transfers.

5 Picks up between 1 to 5 pipette tips, depending on the number of replicates, from the next available column or row of pipette tips in the seating station. Picks up tips in row format for top-to-bottom or bottom-to-top dilution series. Picks up tips in column format for left-to-right or right-to-left dilution.

Protocol step

Head moves to deck location

Action

18 Serial Dilution v3.0 User Guide Assay development guidelines

682 AssayMAP Protein Sample Prep Workbench User Guide

9. Additional Serial Dilution Transfers

6 Moves to the lowest concentration wells of the dilution series in the serial dilution plate. If a blowout volume is specified, aspirates an air gap. If specified, prewets the pipette tips with the sample. Aspirates the sample into the pipette tips, and then dispenses the sample into

the next set of dilution wells in the serial dilution plate. If specified, mixes the well contents for the set number of cycles. If specified, performs a blowout, followed by a tip touch on the east and west

sides of the wells. Repeats steps 7 and 8 until the serial dilution series is complete.

10. Final Tip Ejection

3 Ejects the used pipette tips into the tip box.

11. Park Head

1 Moves to a safe height above the wash station.

Protocol step

Head moves to deck location

Action

683

Emergency stops and pauses

Stopping in an emergency

To stop in an emergency: Press the red button on the emergency-stop pendant. The Bravo head stops immediately.

The robot disable is active error message opens.

To restore the Bravo device after an emergency stop: 1 At the emergency-stop pendant, turn the red button clockwise. The spring-

loaded button pops up.

2 If applicable. Remove any object that is interrupting the Light Curtain. 3 In the AssayMAP Bravo Error dialog box, click Retry.

In most cases, the AssayMAP Bravo Platform will be able to resume the run where it left off.

19 Emergency stops and pauses

684 AssayMAP Protein Sample Prep Workbench User Guide

Pausing a run

To pause and then continue a run: 1 In the workbench application or utility control panel, click Pause.

The task currently in progress finishes before the protocol pauses. The Scheduler Paused dialog box opens.

2 While the Bravo device is idle, make the necessary changes to your run, for example, adjusting a labware position or volume.

3 To resume the run, click Continue in the Scheduler Paused dialog box. To cancel the run, click Abort process in the Scheduler Paused dialog box.

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