Protocol Guide Original Instructions

[Pages:29]AssayMAP Peptide Cleanup

Protocol Guide

Original Instructions

Notices

? Agilent Technologies, Inc. 2013

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.

Manual Part Number

G5409-90013

Edition

Revision 01, July 2013

Contact Information

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

Technical Support: 1.800.979.4811 (US only) or +1.408.345.8011 (rest of the world) service.automation@

Customer Service: 1.866.428.9811 or +1.408.345.8356 orders.automation@

European Service: +44 (0)8457125292 euroservice.automation@

Documentation feedback: documentation.automation@

Web: lifesciences/ automation

Acknowledgements

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

Warranty

Safety Notices

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 warranties, 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 contained 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 agreement shall control.

WARNING

A WARNING notice denotes a hazard. 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 indicated conditions are fully understood and met.

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 nonDOD Departments and Agencies of the U.S. Government will receive no greater than Restricted Rights as defined in FAR 52.22719(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.

CAUTION

A CAUTION notice denotes a hazard. 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 proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

AssayMAP Peptide Cleanup Protocol Guide

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Contents

Preface ....................................................................................................................................................................................................... 4 About this guide....................................................................................................................................................................... 4 Reporting problems ................................................................................................................................................................. 4

Before you begin ...................................................................................................................................................................................... 5 Equipment ................................................................................................................................................................................. 5 Kits and labware requirements ............................................................................................................................................. 7 Reagent requirements ............................................................................................................................................................ 7 Cartridge use and storage guidelines .................................................................................................................................. 8

Preparing your samples........................................................................................................................................................................... 9 Sample particulates................................................................................................................................................................. 9 Sample composition................................................................................................................................................................ 9 Sample load volume ................................................................................................................................................................ 9 Sample plate setup................................................................................................................................................................ 10

Preparing your reagents........................................................................................................................................................................ 11 Using the Peptide Cleanup Calculator ............................................................................................................................... 11 Preparing reagents for the Peptide Cleanup protocol .................................................................................................... 13

Setting up a Protocol ............................................................................................................................................................................. 14 Starting up............................................................................................................................................................................... 14 Installing software and opening the application interface ............................................................................................ 14 Opening the protocol and app ............................................................................................................................................. 14 Priming the wash station and initializing the Bravo platform ....................................................................................... 16

Running a protocol ................................................................................................................................................................................. 18 Assay Development guidelines............................................................................................................................................................ 22 Automation Protocol Steps .................................................................................................................................................................. 28

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Preface

About this guide

This guide is designed for both routine operators and assay developers. This guide does not cover how to set up and maintain the Bravo Platform, detailed safety guidelines, details about the Bravo 96AM Head, details about the Pump Module, or detailed editing of the protocol or application interface in the VWorks software. For information on these topics, go to the Automation Solutions knowledge base at chem/askb, and click the link for PDF user guides.

Reporting problems

If you find a problem with the AssayMAP Peptide Cleanup solution, contact Automation Solutions Technical Support. For contact information, see Notices on page ii.

For reporting problems with... Have the following information ready...

Hardware

Device serial number from the Bravo serial number label

Software

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

control software, diagnostics software, and firmware) Error message Relevant files, such as log files

Protocol Guide

Send your comments in an email to documentation@

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Before you begin

Introduction

The AssayMAP Peptide Cleanup solution enables automated cleanup of 8 to 96 peptide samples in a single run. The solution consists of the following components:

Agilent Bravo Platform for AssayMAP (liquid handler) Agilent AssayMAP microchromatography cartridges packed with either a 20 m diameter silica resin with 150 ? pores derivitized with C18 (C18) or a 20 m diameter underivitized polystyrene polystyrene hydrophobic reversed-phase resin with 100 ? pores (RP-S). Agilent software and supporting files (VWorks software, Launch Pad, AssayMAP Peptide Cleanup protocol, and application interface)

Equipment

Table AssayMAP Bravo Platform requirements

Component

Bravo Platform for AssayMAP technology Includes the base Bravo Platform, computer, and the following:

Gripper Upgrade Bravo 96AM Head 96AM Wash Station Pump Module 2.0 96 AM Cartridge & Tip Seating Station

Custom Plate Nest Risers, 146 mm Peltier Thermal Station with STC controller Orbital Shaking Station with Control Unit Red PCR Plate Insert Two 10-L carboys

Part number

G5542A

16545-101 G5498B#046 G5498B#057 G5498B#058 G5409-20025

G5498B#017 G5498B#055 G5498B#035 G5498B#033 G5498B#013 NA

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Figure AssayMAP Bravo Platform and accessories

Table Optional Equipment

Centrifuge with microplate rotor SpeedVac PlateLoc

Software

This guide documents the following versions, which are required for the Bravo 96AM Head: VWorks Automation Control 11.4.0.1233 Bravo Diagnostics 17.1.0.1 Bravo Firmware 5.4.7 AssayMAP Launch Pad v3.0

NOTE

You can download the most recent version of the installer from lifesciences/ambravodownloads

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Kits and labware requirements

RP-S and C18 reverse phase cartridge types are currently available for performing peptide cleanup on the AssayMAP Bravo. Each of these cartridge types can either be purchased as a rack of 96 cartridges plus the labware required for peptide cleanup and in solution digestion or as an individual rack of 96 cartridges.

Table AssayMAP Digestion and Cleanup Starter kits

AssayMAP Digestion and RP-S G5496-60034 Cleanup Starter Kit

AssayMAP Digestion and C18 Cleanup Starter Kit

G5496-60013

96 reversed-phase (RP-S) cartridges plus labware for InSolution Digestion and Peptide Cleanup

96 C18 cartridges plus labware for In-Solution Digestion and Peptide Cleanup

AssayMAP cartridge racks*

5190-6532 G5496-60033

AssayMAP C18 cartridge rack (Qty 96) AssayMAP RP-S cartridge rack (Qty 96)

* Labware is included in Starter Kits, but required for In-Solution DigestionA and Peptide CleanupB when using individual racks.

2 1.2mL Deep-Well Plates (AbGene AB-1127)A 11 96-Well Round Bottom, White Plates (Greiner 650207)A 1 250 ?L Pipette Tips (Agilent 19477-02)A 4 12-Column Low-Profile Reservoirs (Seahorse 201280-100)B 2 96-Well Round-Bottom, Clear Plates (Greiner 650201)B 3 96-Well PCR Plates (Eppendorf 30129300)A,B

CAUTION

The supplied protocol and app support a specific list of labware only. Using undefined or incorrectly identified labware on the Bravo deck can cause a crash and potential damage to the Bravo 96AM Head. For more details contact technical support.

Reagent requirements

The simplest deck configuration for the Peptide Cleanup has three solvents on the Bravo deck: 50% ACN:50% H2O:0.1%TFA, 100% H2O:0.1%TFA, and deionized water Table Required solvents to be supplied by customer

Solution

Quality

Estimated Volume*

acetonitrile (ACN)

100%, HPLC-grade

~100 mL

trifluoroacetic acid (TFA)

100%, HPLC-grade

~0.3 mL

purified deionized water (H2O) HPLC-grade, MilliQ, or equivalent ~5 L * Use provided Reagent Calculator for more accurate volumes and suggested overages.

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Cartridge use and storage guidelines

Follow these guidelines to get the best performance from the AssayMAP cartridges:

Use only particulate-free samples, from a previous chromatographic run or by filtration (0.22 m).

Use only primed and equilibrated cartridges. We guarantee performance when you use AssayMAP cartridges on the same day you equilibrate them. Do not allow wetted cartridges to dry out. We do not guarantee performance of stored cartridges following equilibration.

Store unused cartridges in the original shipping package at room temperature. Cartridges stored in this manner are stable for 6 months from the date of purchase.

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