ProteOn XPR36 User manual

ProteOn™XPR36
Protein Interaction Array System
Protein Interaction Analysis
User Manual

ProteOn XPR36
Protein
Interaction
Array System
Version 3.1 User Manual
™
™

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BIO-RAD TECHNICAL SUPPORT
Bio-Rad Technical Support in the United States is open Monday-Friday,
5:00 am to 5:00 pm, Pacific Standard Time.
Phone: [phone] (U.S. and Canada Only)
Fax: [phone]
E-mail: [e-mail] (USA and Canada Only)
Visit us on the Web at discover.bio-rad.com.
For international customers, contact your local Bio-Rad office. Contact
information for the Bio-Rad offices is available on the Web and on the back
cover of this manual.
Worldwide technical support is available on the Web at consult.bio-rad.com.
NOTICES
No part of this publication may be reproduced or transmitted in any form or by
any means, electronic or mechanical, including photocopying, recording, or
any information storage or retrieval system, without permission in writing from
Bio-Rad.
When running ProteOn Manager™ software in regions of the world where
decimals are expressed as commas, an error reading floating point values
might occur. Please use U.S. English numeric and date formats, as set in the
Control Panel, to avoid this problem. ProteOn Manager software has been
certified to run only using U.S. English.
TRADEMARKS
Adobe is a trademark of Adobe Systems Incorporated. Excel, Windows,
Windows Server, and Windows XP are trademarks of Microsoft Corporation.
The MySQL database is licensed from and owned by MySQL AB, a subsidiary
of Sun Microsystems, Inc. The program 7-Zip is used to extract database
files. It is licensed under a GNU LGPL license. You may find the source code
and other information at www.7-zip.org. NeutrAvidin is a trademark of Pierce
Biotechnology, Inc. Contrad is a trademark of Decon Laboratories. Kimwipes
is a trademark of Kimberly-Clark Corporation. Tween is a trademark of ICI
Americas Inc. HASP is a trademark of SafeNET Inc.

ProteOn XPR36 System |
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LIMITATIONS OF LIABILITY
Bio-Rad is not responsible for the misinterpretation of results obtained by
following the instructions in this manual. Whenever possible, you should
contact the Technical Support Department at Bio-Rad to discuss your results.
As with all scientific studies, we recommend that you repeat your experiment
at least once before making any significant conclusions for presentation or
publication.
Copyright © 2011 by Bio-Rad Laboratories, Inc. All rights reserved.
REFERENCES
Antibody characterization and development using the ProteOn™ XPR36
protein interaction array system, Bio-Rad Bulletin 5412 (2007)
Bravman T et al. (2006). Exploring 'One-shot' Kinetics™ and small-molecule
analysis using the ProteOn™ XPR36 array biosensor, Analytical Biochemistry,
Vol. 358, Issue 2, pp. 281–288.
Bravman T et al. (2007). Screening, ranking, and epitope mapping of anti-
human IL–9 supernatants, Bio-Rad Bulletin 5540.
Bravman T et al. (2008). The ProteOn™ XPR36 array system – high throughput
kinetic binding analysis of biomolecular interactions, Cellular and Molecular
Bioengineering, Vol. 1, pp. 216–228.
Bronner V et al. (2010). Rapid assay development and optimization for small
molecule drug discovery, Bio-Rad Bulletin 5797.
Bronner V et al. (2009). Rapid screening and selection of optimal antibody
capturing agents using the ProteOn™ XPR36 protein interaction array system,
Bio-Rad Bulletin 5820.
Bronner V et al. (2010). Therapeutic antibodies: Discovery and development
using the ProteOn XPR36 biosensor interaction array system, Analytical
Biochemistry, Vol. 406, Issue 2, pp. 147–156.
Bronner V et al. (2006). Rapid and efficient determination of kinetic rate
constants using the ProteOn™ XPR36 protein interaction array system, Bio-
Rad Bulletin 3172.
Bronner V et al. (2006). Mechanisms of protein-protein binding: double mutant
cycle analysis using the ProteOn™ XPR36 system, Bio-Rad Bulletin 5358.
Bronner V et al. (2006). Rapid and detailed analysis of multiple antigen-
antibody pairs using the ProteOn™ XPR36 protein interaction array system,
Bio-Rad Bulletin 5360.
Bronner V et al. (2006). Rapid optimization of immobilization and binding
conditions for kinetic analysis of protein-protein interactions using the
ProteOn™ XPR36 protein interaction array system, Bio-Rad Bulletin 5367.

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Bronner V et al. (2006). Analysis of multiple protein-protein interactions using
the ProteOn™ XPR36 protein interaction array system, Bio-Rad Bulletin 5368.
Cohen S et al. (2010). Applications of the ProteOn™ NLC sensor chip:
antibody-antigen, DNA-protein, and protein-protein interactions, Bio-Rad
Bulletin 5449.
Moriarty L. (2010). Guide to ligand immobilization on the ProteOn™ XPR36
system, Bio-Rad Bulletin 5821.
Nahshol O et al. (2008). Parallel kinetic analysis and affinity determination of
hundreds of monoclonal antibodies using the ProteOn™ XPR36, Analytical
Biochemistry, Vol. 383, Issue 2, pp. 52–60.
Stephen A et al. (2009). Determining the binding kinetics of HIV–1
nucleocapsid protein to six densities of oligonucleotide using the ProteOn™
XPR36 protein interaction array system, Bio-Rad Bulletin 5846.
Tabul M et al. (2010). Rapid, high-throughput screening of protein kinase
inhibitors using the ProteOn™ XPR36 protein interaction array system, Bio-
Rad Bulletin 5965.
Turner B et al. (2008). Applications of the ProteOn™ GLH sensor chip:
interactions between proteins and small molecules, Bio-Rad Bulletin 5679.
SAFETY
Your safety is important to us. The ProteOn™ XPR36 protein interaction array
system contains components that, if handled improperly, are potentially
harmful. The protection provided by the equipment can be compromised or
the warranty voided if the equipment is used in a manner not specified by
Bio-Rad Laboratories. Bio-Rad recommends that all system users become
familiar with the comprehensive operating procedures and safety information
in this manual. This information alerts you to potential hazards. Please review
all safety information and adhere to standard laboratory safety practices.
WARNING NOTICES USED IN THIS MANUAL
The triangle warning symbol indicates the possibility of personal
injury or damage to the equipment if instructions are not followed.
ELECTRICAL SAFETY
The instrument must be connected only to an approved power
source. Use a grounded socket that meets power requirements. For
more information, refer to Appendix B Specifications and
Requirements on page 209.
Do not attempt to clean the instrument’s electrical components.

ProteOn XPR36 System |
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In the event of a fluid leak, turn off all power to the system and disconnect all
power cords. Contact Bio-Rad Laboratories for further information.
UNINTERRUPTIBLE POWER SUPPLY
An uninterruptible power supply is recommended to protect the ProteOn
XPR36 from an unstable main power source, harmful spikes, surges or total
blackouts.
This table contains minimum specifications for an uninterruptible power
supply that can be used with the ProteOn instrument.
MECHANICAL SAFETY
Use caution when handling the needle tips; they are sharp.
Keep fingers clear of the autosampler during operation.
Shut down the instrument and turn off the power before handling
the autosampler arm, needle holder, or needles.
BIOLOGICAL SAFETY
All human and animal samples may contain hazardous infectious
agents. Follow appropriate biosafety procedures when handling
these products and their containers.
Observe all local, state, and federal biohazard handling
regulations when disposing of biohazardous waste material.
Specification 120 VAC 230 VAC
Output power capacity 1,500 VA 1,500 VA
Nominal input voltage 120 V 230 V
Backup time Minimum 15 min at full load Minimum 15 min at full load

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PERSONAL SAFETY
The ProteOn XPR36 instrument weighs 187 lb (85 kg).
Follow all personal safety precautions when moving the
instrument.
If you must lift the instrument, use proper lifting techniques. Have two people
lift the instrument, and use the lifting points indicated below:.
Caution: Bio-Rad strongly recommends that you keep your
boxes for future moving and hire a professional service to safely
move the instrument.

ProteOn XPR36 System |
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Contents
Chapter 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Experiment Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
ProteOn Manager Software Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Software Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Instrument Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Quick Start Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Toolbar Icons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
ProteOn Manager Software Options . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Database Browser Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Start Menu Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Instrument Shutdown Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Instrument Log Extraction Utility . . . . . . . . . . . . . . . . . . . . . . . . 9
Backing Up the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Installing ProteOn Manager Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Installing ProteOn Manager 3.1 Software . . . . . . . . . . . . . . . . . . . . . . . 11
Upgrading From Earlier Versions (v. 2.1.n, v. 3.0, or v. 3.0.1) to v. 3.1 14
Optimizing the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Reinstalling Version 3.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Importing Example Chip Conditioning Protocols . . . . . . . . . . . . . . . . . 19
Importing Files from Another Computer . . . . . . . . . . . . . . . . . . . . . . . . 19
Saving/Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Importing Data from Previous Versions . . . . . . . . . . . . . . . . . . . . . . . . . 20
Starting and Exiting ProteOn Manager Software . . . . . . . . . . . . . . . . . . . . . . . 21
Starting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Exiting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
ProteOn XPR36 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Starting the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
System Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Component Status LEDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Status Bar Instrument State Messages . . . . . . . . . . . . . . . . . . . . . . . . . 23
Inactive State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Power Up State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Initializing State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Ready State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Running State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Stop State to Ready State . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

ProteOn XPR36 System | Contents
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Standby State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Maintenance State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Maintenance Standby State . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Shutting Down State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Fault State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Flushing State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Buffer System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Flushing and Priming the System . . . . . . . . . . . . . . . . . . . . . . . 27
Selecting Buffer Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Chapter 2. Protocol Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Creating and Saving Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Creating a Protocol from Rack/Plate Contents . . . . . . . . . . . . 35
Creating Sample Data From a File . . . . . . . . . . . . . . . . . . . . . . 37
Configuring Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Editing Protocol Sample Details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Importing and Adding Racks/Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Creating Protocol Steps from Sample Panels . . . . . . . . . . . . . . . . . . . 44
Sample Panel Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Changing Sample Positions or Copying Samples . . . . . . . . . . . . . . . . 46
Autosampler Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Creating and Editing Protocol Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Setting Injection Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Protocol Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Copying and Pasting Protocol Steps . . . . . . . . . . . . . . . . . . . 51
Editing Injection Step Details. . . . . . . . . . . . . . . . . . . . . . . . . . 52
Setting the Buffer Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Setting the Temperature Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Setting the Change Rack Step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Setting a Pause Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Protocol Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Protocol and Sample Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Chapter 3. Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Experiment Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Dissolving and Aliquoting ProteOn EDAC and Sulfo-NHS . . . . . . . . . . 64
Preparing the Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Loading the Buffer Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Priming the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

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Flushing the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Preparing the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Setting the Autosampler Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 69
Sensor Chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Sensor Chip Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Storage and Temperature Equilibration . . . . . . . . . . . . . . . . . . . . . . . . 72
Opening the Sensor Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Conditioning the Sensor Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
GLC, GLM, and GLH Sensor Chip Conditioning. . . . . . . . . . . 73
NLC Sensor Chip Conditioning . . . . . . . . . . . . . . . . . . . . . . . . 75
HTG Sensor Chip Conditioning . . . . . . . . . . . . . . . . . . . . . . . . 75
Initializing the Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Inserting and Ejecting the Sensor Chip . . . . . . . . . . . . . . . . . . . . . . . . 76
Chip Loader LEDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Setting Sensor Chip Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Reusing Chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Viewing Chip Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Running Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Nonspecific Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Experiments with Highly Refractive Cosolvents (DMSO) . . . . . . . . . . . 84
Saving and Exporting Experiment Data . . . . . . . . . . . . . . . . . . . . . . . . 86
Managing Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Chapter 4. Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Tools for Viewing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Sensorgram Data Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Changing Sensorgram Appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Changing Sensorgram Appearance in a Dataset . . . . . . . . . . . 92
Changing Sensorgram Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Showing Interspot Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Showing Point Minimized Data . . . . . . . . . . . . . . . . . . . . . . . 95
Showing Kinetic Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Viewing Kinetic Data in an Isoaffinity Graph . . . . . . . . . . . . . . 96
Visually Comparing Tabular Data in Screening Graphs . . . . . 97
Excluding Sensorgrams From Manual Processing . . . . . . . . . 99
Comparing Data in Multiple Sensorgram Data Windows . . . . 100
Undo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Redo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Copying Sensorgram Data Windows . . . . . . . . . . . . . . . . . . . . . . . . . 101
Tabulated Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101
Data Filters and Display Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

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Choosing a Panel Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Selecting a Protocol Step for Analysis . . . . . . . . . . . . . . . . . . 103
Grouping Data for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Interaction Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Display Control Grid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Processing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Selecting a Range on a Sensorgram . . . . . . . . . . . . . . . . . . . 108
Data Processing Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Using the Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109
Selecting all Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Copying Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Processing Multiple Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Aligning the Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .110
Aligning the Baseline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Removing Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Moving a Sensorgram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Performing Reference Subtraction . . . . . . . . . . . . . . . . . . . . . 111
Displaying Real-Time Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Deleting Report Point and User-Defined Columns . . . . . . . . . 113
Displaying and Hiding Markers . . . . . . . . . . . . . . . . . . . . . . . . 113
Remove Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Switching to Raw Dataset . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Processing EV Correction Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Working With EVC Calibration Plots . . . . . . . . . . . . . . . . . . . . 117
Creating a Dataset . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .118
Renaming a Dataset . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .118
Deleting a Dataset . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Analyzing Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Analysis Datasets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Parameter Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Specifying Report Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Specifying Report Points on Concatenated Data . . . . . . . . . 122
Computing Difference Between Report Points . . . . . . . . . . . 122
Viewing Report Points from a Dataset . . . . . . . . . . . . . . . . . . 123
Importing Report Points from Other Datasets . . . . . . . . . . . . 123
Saving Report Points to an External File . . . . . . . . . . . . . . . . 124
Adding a Report Points Column . . . . . . . . . . . . . . . . . . . . . . . 124
Deleting Report Point Columns . . . . . . . . . . . . . . . . . . . . . . . 124
Filtering and Sorting Analysis Results . . . . . . . . . . . . . . . . . .124
Adding a User-Defined Column . . . . . . . . . . . . . . . . . . . . . . . 124
Deleting a User-Defined Column . . . . . . . . . . . . . . . . . . . . . . 125
Renaming an Analysis Set . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

xiii
Deleting an Analysis Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Analysis Wizards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Kinetic Analysis Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Equilibrium Analysis Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Concentration Analysis Wizard . . . . . . . . . . . . . . . . . . . . . . . . 134
Ungrouping Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Filtering Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Sorting Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Saving and Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Exporting Data to a File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Exporting Analysis Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Copying and Pasting Data to a File . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Exporting Experiments to a File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Customizing and Printing Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Exporting a Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Chapter 5. Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Maintenance and Cleaning Chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Maintenance Chips (MNT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Cleaning Chips (CLN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Instrument Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151
Scheduled Maintenance Wizards . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Maintenance Protocol Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Maintenance Wizard Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Prime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Weekly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Postexperiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Clean MCM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Syringe Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .156
Starting the Syringe Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Cleaning Syringes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Replacing the Syringes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Resuming the Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
OQ Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .165
Shutdown Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Immediate Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Long-Term Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Software-Only Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Exiting ProteOn Manager Software and the Instrument . . . . . . . . . . . 168

ProteOn XPR36 System | Contents
xiv
Emptying the Collection Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Chapter 6. ProteOn System Troubleshooting . . . . . . . . . . . . 171
Common Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Nonspecific Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Addressing the Causes of NSB . . . . . . . . . . . . . . . . . . . . . . . 173
Firewall Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
High Throughput Screening Analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Chapter 7. Security Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Background on U.S. FDA Part 11 Regulations (21 CFR Part 11) . . . .177
Standard Mode vs. Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Installing and Starting ProteOn Manager Software, Security Edition. 178
Enabling and Disabling Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . 179
Users, Passwords, and User Levels . . . . . . . . . . . . . . . . . . . . . . . . . . 180
User Level Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
User Access by Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
User Authentication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
File Security and Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Importing/Exporting Secure Files . . . . . . . . . . . . . . . . . . . . . . 182
ProteOn Manager Software Logs. . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Secure Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Signed Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Signed Datasets and Analysis Sets . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Viewing the Audit Trail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Protected Directories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Logging Off. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Changing Data Ownership . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
A. ProteOn XPR36 Instrument and Peripherals . . . . . . . . . . 189
Instrument Front View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Instrument Side View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Instrument LEDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192
Chip Loader and Chip Eject Button . . . . . . . . . . . . . . . . . . . . . . . . . .193
Buffer Control Buttons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Serial Numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Cables and Power Switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

xv
Ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Inside the Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Buffer System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Microfluidics System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Thermal Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Needle Holder with Sampling Needles . . . . . . . . . . . . . . . . . . . . . . . . 200
Changing the Needle Holder and Needles . . . . . . . . . . . . . . . . . . . . .200
Wash Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Sample Holders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Sample Holder Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Syringe Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Multichannel Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Optical Detection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Reservoir, Overflow Sensor, and Drain Pump . . . . . . . . . . . . . . . . . . .206
Computer and Display. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
HASP Keys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
B. Specifications and Requirements . . . . . . . . . . . . . . . . . . . 209
System Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Detection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Detection Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Working Ranges (Typical Values) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Physical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Installation Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Electrical Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Bench Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Environmental Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Chemical Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
C. Surface Plasmon Resonance. . . . . . . . . . . . . . . . . . . . . . . 213
Biomolecular Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Interaction Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Ligand Immobilization Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Analyte Interaction Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Ligand Immobilization Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Immobilization Chemistries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Direct, Covalent Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . 215

ProteOn XPR36 System | Contents
xvi
Noncovalent Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Activation Step (Amine Coupling). . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Immobilization Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Determining the Optimum pH for Immobilization . . . . . . . . . . . . . . . . . . . . . . 219
Deactivation and Stabilization Steps . . . . . . . . . . . . . . . . . . . . . . . . . 220
Determining the Extent of Ligand Immobilization . . . . . . . . . . . . . . . 221
Analyte Interaction Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Analyte Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Regeneration Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
D. Protocol Development Kits . . . . . . . . . . . . . . . . . . . . . . . . 225
One-shot Kinetics™ (OSK)™ Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Multiple Protein Interaction Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Protein–Small Molecule Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
E. Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
How to Order. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Payment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .227
Phone-in Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Information Needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Materials and Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
F. Security Edition Configuration Guide . . . . . . . . . . . . . . . . 233
Standard Mode vs. Secure Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Security Edition Hardware Protection Key (HASP Key) . . . . . . . . . . . 233
ProteOn Manager Software Users and Groups . . . . . . . . . . . . . . . . . . . . . . . 234
ProteOn Manager Software User Groups . . . . . . . . . . . . . . . . . . . . . . 234
Configuring Users and Groups on the Local Computer . . . . . . . . . . . . . . . . . 235
Configuring Users and Groups on a Network Domain . . . . . . . . . . . . . . . . . . 239
Windows Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Password Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Password Policy Setting Examples . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Account Lockout Policy Setting Examples . . . . . . . . . . . . . . . . . . . . 243
Auditing Windows Event Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Miscellaneous Security Measures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

ProteOn XPR36 System
1
1Introduction
The ProteOn™ XPR36 protein interaction array system consists of the
ProteOn™ XPR36 instrument, the controller (the computer attached to the
instrument), a HASP key, and ProteOn Manager™ software. This manual
describes how to use the ProteOn XPR36 protein interaction array instrument
with ProteOn Manager software.
This chapter describes the experiment workflow and provides an overview of
ProteOn Manager software. It describes the software layout and tools and
how to upgrade the software and start the ProteOn system. Chapter 7
explains how to use the optional ProteOn Manager software, Security Edition.
This chapter also has instructions for controlling the instrument and explains
the LED indicators, various instrument states, and the buffer control system.
Experiment Workflow
ProteOn Manager software controls the operation, data collection, data
analysis, and maintenance of the ProteOn XPR36 instrument. The software
license permits its installation and use on both the controller and on a
separate PC for offline protocol creation and data analysis.
Tabs in the software navigation panel guide you through the experiment
workflow.
Steps Navigator Tab Navigator Tab Functions
1Configure the
instrument
Instrument Set up the instrument, initialize a sensor chip, set the
autosampler temperature, choose buffers, view the
instrument log, and perform routine maintenance
2Design or edit a
protocol
Protocol Create and edit protocols; define samples, protocol
steps, and sample layout for a protocol; and print the
protocol and sample layouts
3Run and view an
experiment
Run Run an experiment and view the status of an
experiment in progress
4Data processing Data Displays only when an experiment is open; organize
data for processing and analysis and create and view
datasets

ProteOn XPR36 | Introduction
2
ProteOn Manager Software Features
Software feature descriptions and manual pages where they are discussed.
5Analyze the
datasets
Analysis
Datasets
Displays only when an experiment is open; analyze
selected processed datasets and view existing analysis
data
Steps Navigator Tab Navigator Tab Functions
Quick Start Menu
Rapid navigation to commonly performed tasks page 6
Instrument Control
Log continuously records instrument and application events page 4
Maintenance wizards and protocols page 152
MCM (multichannel module) maintenance procedure wizard page 155
Protocol Design
Predesigned, application-based protocol templates assist with experiment design page 32
Guided stepwise protocol setup page 44
Protocol Sample Editor
Easy sample entry and viewing; import page 42
Assign samples to racks or microplates page 44
Sample layout screen displays rack/microplate position and required volume for each sample page 61
Sample layout report can be printed and used as a guide to fill racks or microplates page 61
Protocol Step Editor
Drag-and-drop protocol step editor for easy protocol creation page 50
Drag and drop commonly used step groups for organization and rapid protocol creation page 51
Graphical interaction layout to guide experiment setup page 52
Data Controls
Data viewing, organization, and processing controls page 89
Sample panel chooser filters datasets by type of sample: activator, ligand, deactivator,
regenerator, analyte, blank, and excluded volume (EV) correction calibration standard
page 45
Protocol step chooser selects protocol steps from which data are viewed page 48
Data grouping chooser organizes data into groups for display, processing, and analysis page 103
Interaction display chooser to select, view, and hide interaction data (sensorgrams) and their
sample information
page 107