Criterion Precast Gels Instruction Manual and Application Guide ™

Criterion Precast Gels Instruction Manual and Application Guide ™
Criterion Precast Gels
™
Instruction Manual and Application Guide
Bio-Rad Technical Support
For help and technical advice, please contact the Bio-Rad Technical Support department. In the United States, the Technical
Support department is open Monday–Friday, 5:00 am–5:00 pm, Pacific Time.
Phone: 1-800-424-6723
Fax: 1-510-741-5802
Email: [email protected] (for U.S. and international customers)
Online technical support and worldwide contact information are available at www.consult.bio-rad.com.
Legal Notices
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including
photocopy, recording, or any information storage or retrieval system, without permission in writing from Bio-Rad Laboratories.
Bio-Rad reserves the right to modify its products and services at any time. This user guide is subject to change without notice.
Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for any damages resulting from the application
or use of this information.
Brij and Tween are trademarks of ICI Americas, Inc. Coomassie is a trademark of BASF Aktiengesellschaft. Ficoll is a trademark
of Amersham Pharmacia Biotech. StrepTactin is a trademark of Institut für Bioanalytik GmbH. StrepTactin is covered by German
patent application P 19641876.3. Bio-Rad Laboratories, Inc. is licensed by Institut für Bioanalytik GmbH to sell these products
for research use only. SYBR is a trademark of Invitrogen Corporation. SYPRO is a trademark of Molecular Probes, Inc. Bio-Rad
is licensed to sell SYPRO products for research use only, under U.S. Patent 5,616,502. Triton is a trademark of Union Carbide.
Criterion™ TGX Stain-Free™ and Criterion Stain Free™ precast gels are covered by U.S. Patent No. 7,569,130.
Purchase of Criterion™ XT Bis-Tris gels, XT MOPS running buffer, XT MES running buffer, XT MOPS buffer kit, and XT MES buffer
kit is accompanied by a limited license under U.S. patents 6,143,154; 6,096,182; 6,059,948; 5,578,180; 5,922,185; 6,162,338;
and 6,783,651 and corresponding foreign patents.
Copyright © 2011 by Bio-Rad Laboratories. All rights reserved.
Contents
Chapter 1: Criterion™ Precast Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Gel Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Comb Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.4 Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.5 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.6 Important Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chapter 2: Setup and Basic Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1 Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3 Setting Up and Running Criterion Gels in the Criterion Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.4 Removing the Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Chapter 3: SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2 Criterion Gel Selection Guide for SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.1 Criterion™ TGX™ and Criterion™ TGX Stain-Free™ Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2.2 Criterion Tris-HCl and Criterion Stain Free™ Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2.3 Criterion™ XT Bis-Tris Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2.4 Criterion XT Tris-Acetate Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.3 SDS-PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3.1 Running Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3.2 Sample Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Chapter 4: Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2 Criterion Gel Selection Guide for Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2.1 Criterion TGX and Criterion TGX Stain-Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2.2 Criterion Tris-HCl and Criterion Stain Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.2.3 Criterion XT Tris-Acetate Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.3 Native PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Chapter 5: Criterion Stain Free System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5.2 Criterion Stain Free Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.3 Electrophoresis with Criterion TGX Stain-Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.4 Using the Gel Doc™ EZ Imager . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Chapter 6: Peptide Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.2 Criterion Tris-Tricine/Peptide Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.3 Tris-Tricine/Peptide Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
6.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chapter 7: Isoelectric Focusing (IEF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7.2 Criterion IEF Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7.3 IEF Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
7.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Chapter 8: Protease Analysis by Zymogram PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
8.2 Criterion Zymogram Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
8.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
8.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
8.3 Zymogram Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
8.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
8.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Chapter 9: Nondenaturing Nucleic Acid PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
9.2 Criterion TBE Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
9.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
9.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
9.3 Nondenaturing Nucleic Acid PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
9.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
9.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Chapter 10: Denaturing Nucleic Acid PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.2 Criterion TBE-Urea Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.3 TBE-Urea Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
10.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Chapter 11: 2-D Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
11.2 Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
11.3 Agarose Overlay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
11.4 Second-Dimension Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Chapter 12: Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
12.1 SDS-PAGE and Native PAGE Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
12.2 Peptide Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
12.3 IEF Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
12.4 Zymogram Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
12.5 TBE Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
12.6 TBE-Urea Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Chapter 13: Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
13.2 Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
13.2.1 Transfer Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
13.2.2 Wet Transfer Using the Criterion Blotter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
13.2.3 Transfer Using the Trans-Blot® Turbo™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
13.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
13.3 Total Protein Blot Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
13.4 Immunodetection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Chapter 14: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Appendix A: Quick Start Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Appendix B: Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Appendix C: Related Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Appendix D: Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
1
Criterion™ Precast Gels
1.1 Introduction
Criterion precast gels are an effective system for performing polyacrylamide gel electrophoresis (PAGE).
These 13.3 x 8.7 cm gels are wider and longer than traditional mini format gels, and their innovative,
easy-to-use design produces excellent resolution while accommodating more samples per gel.
Designed for use with the Criterion family of vertical electrophoresis cells, which includes the Criterion
(2-gel capacity) and Criterion™ Dodeca™ (12-gel capacity) cells, Criterion precast gels allow separation of
more samples than mini format gels and provide significant cost and time savings. Some of the unique
features provided are:
Integrated buffer chamber that eliminates buffer leaks
n
Capacity for up to 26 samples per gel
n
Compatibility with multichannel pipets (12+2 and 26-well)
n
Outlined and numbered wells that simplify sample loading and identification
n
Patented1 J-foot design that eliminates post-run gel processing steps and improves gel
drying and blotting results
n
Criterion™ TGX Stain-Free™ formulations for rapid gel imaging without staining
n
Integrated buffer
chamber
Gel type, expiration
date, and catalog
and lot numbers
Outlined and
numbered wells
1
U.S. patent 6,093,301.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
1
Criterion Precast Gels
1.2 Gel Formulations
Criterion precast gels are available in a range of formulations for virtually every electrophoresis
application (Table 1.1). All Criterion gels are composed of polyacrylamide with a bisacrylamide
crosslinker, and they are available in a selection of single percentages and gradients.
Table 1.1. Criterion precast gel formulations.
Application
Gel Formulation
Sample Buffer
Running Buffer
SDS-PAGE
Criterion Tris-HCl
Criterion Stain Free
Criterion TGX™
Criterion TGX Stain-Free
Criterion™ XT Bis-Tris
Criterion XT Tris-acetate
Laemmli
Tris/glycine/SDS
XT
XT
XT MOPS or XT MES
XT Tricine
Native PAGE
Criterion Tris-HCl
Criterion Stain Free
Criterion TGX
Criterion TGX Stain-Free
Criterion XT Tris-acetate
Native
Tris/glycine
Peptide analysis
Criterion Tris-Tricine
Tricine
Tris/Tricine/SDS
Isoelectric focusing (IEF)
Criterion IEF
IEF
Anode and cathode buffers
Protease detection
Criterion zymogram
Zymogram
Tris/glycine/SDS
dsDNA separation
Criterion TBE
Nucleic acid
Tris/boric acid/EDTA (TBE)
ssDNA and RNA separation
Criterion TBE-urea
TBE-urea
TBE
1.3 Comb Configurations
Comb Type Well Volume
12+2 well1 45 μl with two 15 μl reference wells
18-well 30 μl
26-well115 μl
Prep+2 well
800 μl with two 15 μl reference wells
IPG+1 well 11 cm ReadyStrip™ IPG strip (450 μl) with one 15 μl reference well
1.4 Specifications
Gel material Polyacrylamide
Gel dimensions (W x L)
13.3 x 8.7 cm
Gel thickness 1.0 mm
Resolving gel height 6.5 cm
Cassette dimensions (W x L) 15.0 x 10.6 cm
Cassette material Styrene copolymer
Comb material Polycarbonate
Running buffer
460 ml (per gel)
1
2
Multichannel pipet compatible.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
1.5 Storage Conditions
Table 1.2. Storage conditions for Criterion precast gels. Store gels flat. Shelf life is from date of manufacture; expiration dates
are printed on the cassettes.
Storage Temperature
Gel Formulation
Shelf Life
Ambient
Criterion XT Bis-Tris
12 months
2–8°C
Criterion TGX
Criterion TGX Stain-Free
Criterion Tris-HCl
Criterion Stain-Free
Criterion XT Tris-acetate
Criterion Tris-Tricine
Criterion IEF
Criterion zymogram
Criterion TBE
Criterion TBE-urea
12 months
12 months
12 weeks
12 weeks
8 months
12 weeks
24 weeks
8 weeks
12 weeks
8 weeks
1.6 Important Notes
Use each Criterion precast gel as soon as possible after removing it from the storage pouch.
Improper storage of Criterion precast gels can produce numerous artifacts.
n
Store gels flat
n
Avoid prolonged storage at temperatures above those recommended
n
Do not freeze gels
n
If you suspect your gels have been stored improperly, discard them
Do not run more than one gel type in the same apparatus at the same time. Different gel percentages
and formulations have different conductivities and different run times.
Use unstained standards with Criterion TGX Stain-Free and Criterion Stain Free gels, as some
prestained standards are not detected by the Gel Doc™ EZ imager. To monitor electrophoresis, use
10 µl of a 1:1 mixture of Precision Plus Protein™ unstained and prestained standards.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
3
2
Setup and Basic
Operation
2.1 Workflow Overview
Prepare Buffers
Prepare sample and running buffers
Prepare Gels and
Assemble Electrophoresis Cell
Prepare and Load Samples
Dilute in sample buffer
Perform Electrophoresis
SDS-PAGE (Chapter 3)
Native PAGE (Chapter 4)
Peptide Analysis (Chapter 6)
Isoelectric Focusing (Chapter 7)
Protease Analysis (Chapter 8)
Nondenaturing Nucleic Acid PAGE (Chapter 9)
Denaturing Nucleic Acid PAGE (Chapter 10)
2-D Electrophoresis (Chapter 11)
Analyze the Separation
(Chapter 12)
Blot the Gels (Optional)
(Chapter 13)
4
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
2.2 Required Materials
Criterion™ precast gels
n
Criterion or Criterion™ Dodeca™cell
n
PowerPac™ Basic or PowerPac HC power supply (or equivalent)
n
Sample buffer
n
Running buffer (460 ml per gel)
n
2.3 Setting Up and Running Criterion Gels in the Criterion Cell
1. Each Criterion gel is packaged in a plastic storage tray. Remove the cover of the tray by lifting the
corner tab and pulling it diagonally across the package. Remove the gel from the package.
2. Remove the comb and rinse the wells with deionized water (diH2O) or running buffer.
3. Remove the tape from the bottom of the cassette by pulling the tab across the gel.
4. Insert the cassette into one of the slots in the Criterion cell tank so that the integrated upper buffer
chamber faces the center of the cell (A).
5. Fill each integrated upper buffer chamber with 60 ml
running buffer.
A
Integrated upper buffer
chamber
6. Fill each half of the lower buffer tank with 400 ml
running buffer to the marked fill line.
7. Load samples using a syringe or a pipet with
gel-loading tips.
Optional: place a sample loading guide on the
outer edge of the cassette to help align pipet tips
with the wells (this is particularly useful when using
multichannel pipets).
8. Place the lid on the tank, aligning the color-coded
banana plugs with corresponding jacks on the lid.
See Chapters 3–10 for power supply settings.
2.4 Removing the Gel
1. After electrophoresis is complete, turn off the power
supply and disconnect the electrical leads.
B
2. Remove the lid from the tank and remove the
gel(s) from the cell. Pour off and discard the upper
running buffer.
3. Invert the cassette and place the integral buffer
chamber over the cassette-opening tool built into
the Criterion cell lid (B).
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
5
Criterion Precast Gels
4. Press down firmly to break the seals on both sides of
the cassette. The cassette splits open approximately ¹/3
of the way. Alternatively, open the gel cassette by sliding
the tapered back of the comb into the slits on either
side of the cassette.
C
5. Pull the two halves of the cassette apart from the top to
completely expose the gel (C).
6. Remove the gel by either floating the gel into a fixing or
staining solution or by carefully lifting the bottom edge
of the gel from the cassette.
6
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
3
SDS-PAGE
3.1 Introduction
Criterion™ precast gels provide versatile systems for the separation of proteins by either molecular
weight (SDS-PAGE) or mass-to-charge ratio (native PAGE). (See Chapter 4 for native PAGE applications
and protocols.) This versatility is possible because Criterion gels are made without SDS, allowing the
sample buffer and running buffer to determine the separation mechanism.
SDS-PAGE relies on a discontinuous buffer system. Two ions differing in electrophoretic mobility form
a moving boundary when voltage is applied. Proteins have an intermediate mobility that causes them
to concentrate, or stack, into a narrow zone at the beginning of electrophoresis. As that zone moves
through the gel, the sieving effect of the polyacrylamide gel matrix causes proteins of different molecular
weights to move at different rates. This stacking effect is responsible for the high resolving power of
SDS-PAGE: the sample is loaded in a relatively broad zone, and the moving boundary concentrates the
proteins into sharp bands prior to separation.
Protein samples for SDS-PAGE are prepared using SDS and usually a thiol reducing agent such as
β-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins, giving them a rodlike
shape and similar mass-to-charge ratio. The reducing agent disrupts disulfide bonds between and
within proteins, allowing complete denaturation and dissociation. Heat treatment in the presence of
SDS and reducing agent effectively eliminates the effects of native charge and higher order structure on
electrophoretic mobility, so the migration distance depends primarily on molecular weight.
Molecular weight is estimated by plotting the logarithm of protein molecular weight vs. the relative
mobility (Rf ) of the protein (Rf = distance migrated by the protein/distance migrated by the dye front)
or by using the point-to-point semilog interpolation method in Quantity One® or Image Lab™ software.
Refer to bulletins 3133 and 3144 for more information.
3.2 Criterion Gel Selection Guide for SDS-PAGE
A number of Criterion gel types are available for SDS-PAGE (Table 3.1) in both single and gradient
polyacrylamide percentages. Use the protein migration charts and tables to select the gel type that
offers optimum resolution of your sample:
Use single-percentage gels to separate bands of similar molecular weight. Optimum
separation occurs in the lower half of the gel, so use a percentage in which the protein
migrates to the lower half of the gel
n
Use gradient gels to separate samples containing a broad range of molecular weights.
Gradient gels allow resolution of both high and low molecular weight bands on the same
gel. Larger pore sizes at the top of the gel permit resolution of larger molecules, and smaller
pore sizes toward the bottom of the gel restrict excessive separation of small molecules
n
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
7
Criterion Precast Gels
Table 3.1. Criterion precast gels for SDS-PAGE.
Gel Formulation
Gels
TGX (Laemmli-like)
Criterion™
Description
TGX™
Laemmli-like, extended shelf life gels
Criterion™ TGX Stain-Free™
Laemmli-like, extended shelf life gels with trihalo compounds for rapid fluorescence detection
Tris-HCl (Laemmli)
Tris-HCl Laemmli gels
Criterion Tris-HCl
Criterion Stain Free™
Tris-HCl Laemmli gels with trihalo compounds for rapid fluorescence detection
Bis-TrisCriterion™ XT Bis-Tris
Based on a Bis-Tris HCl buffer system (pH 6.4); use these gels with Criterion XT MES buffer for optimum resolution of small proteins
Bis-Tris
Criterion XT Bis-Tris
Based on a Bis-Tris HCl buffer system (pH 6.4); use these gels with Criterion XT MOPS buffer for optimum resolution of midsized proteins
Tris-acetate
Based on a Tris-acetate buffer system (pH 7.0)
Criterion XT Tris-acetate
3.2.1 Criterion TGX and Criterion TGX Stain-Free Gels
Criterion TGX (Tris-Glycine eXtended shelf life) gels are Laemmli-like gels with a proprietary modification
that extends their shelf life to 12 months and enhances separation characteristics relative to conventional
gel types. The TGX formulation yields Laemmli-like separation patterns with short run times and
exceptionally straight lanes and sharp bands. TGX gels offer excellent staining quality, greater transfer
efficiency, and molecular weight estimation without the need for special, expensive buffers.
These gels are run using standard Laemmli sample buffer and Tris/glycine/SDS running buffer. Two
types of TGX formulations are available:
Criterion TGX — Laemmli-like, extended shelf life gels
n
Criterion TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalo compounds
that allow rapid fluorescent detection of proteins with the Criterion Stain Free system,
eliminating staining and destaining steps for faster results (see Chapter 5 for more details)
n
Gel Composition
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Shelf life ~12 months at 2–8°C; expiration date is printed on each cassette
Gel Percentage 40–200 kD
10% 30–150 kD
12% 20–120 kD
18% 10–50 kD
4–15% 20–250 kD
4–20% 10–200 kD
8–16% 20–120 kD
10–20% 10–100 kD
Any kD™ 1
10–200 kD
1Any
8
Optimum Separation Range
7.5% kD is a unique single-percentage formulation that provides a broad separation range and short running time.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
3.2.2 Criterion Tris-HCl and Criterion Stain Free Gels
These Tris-HCl, Laemmli gels use discontinuous glycinate and chloride ion fronts to form moving
boundaries to stack and then separate denatured proteins by size. They are run using standard Laemmli
sample buffer and Tris/glycine/SDS running buffer.
Criterion Tris-HCl — Tris-HCl formulation that offer a shelf life of 12 weeks
n
Criterion Stain Free — Tris-HCl gels with unique trihalo compounds that allow rapid
fluorescent detection of proteins with the Criterion Stain Free system, eliminating staining
and destaining steps for faster results (see Chapter 5 for more details)
n
Gel Composition
Gel buffer 0.375 M Tris-HCl, pH 8.6
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.6
Shelf life ~12 weeks at 2–8°C; expiration date is printed on each cassette
Gel Percentage Optimum Separation Range
5%
100–250 kD
7.5% 40–200 kD
10% 30–150 kD
12.5% 20–120 kD
15% 10–100 kD
18% 10–50 kD
4–15% 20–250 kD
4–20% 10–200 kD
8–16% 20–120 kD
10–20% 10–100 kD
10.5–14% 25–200 kD
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
9
Criterion Precast Gels
Migration charts for protein standards on Criterion Tris-HCl, Criterion TGX, and TGX Stain-Free gels.
3.2.3 Criterion XT Bis-Tris Gels
Criterion XT Bis-Tris gels are based on a Bis-Tris HCl buffer system (pH 6.4) that uses discontinuous
chloride and MES or MOPS ion fronts to form moving boundaries that stack and separate denatured
proteins by size. This chemistry of XT Bis-Tris gels allows maximum stability and consistent results with
a shelf life of at least 12 months.
Running XT Bis-Tris gels with either XT MES or XT MOPS denaturing running buffer produces different
migration patterns. A combination of these two running buffers and three XT Bis-Tris gels can generate
up to six different migration patterns for small to midsize proteins.
Gel Composition
10
Gel buffer Bis-Tris HCl, pH 6.4
Crosslinker 5% C
Stacking gel 4% T, 5% C
Storage buffer Bis-Tris HCl, pH 6.4
Shelf life 12 months at ambient temperature; expiration date is printed on each cassette
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Gel Percentage Optimum Separation Range
XT MES Buffer
XT MOPS Buffer
10% 12%
4–12% 2.5–200 kD
14–220 kD
1–30 kD
6–66 kD
2.5–200 kD
10–300 kD
Migration charts for protein standards on Criterion XT Bis-Tris gels.
3.2.4 Criterion XT Tris-Acetate Gels
Criterion XT Tris-acetate gels are based on a Tris-acetate buffer system (pH 7.0). It uses discontinuous
acetate and Tricine ion fronts to form moving boundaries that stack and separate large denatured
proteins by molecular weight.
Gel Composition
Gel buffer Tris-acetate, pH 7.0
Crosslinker 3.8% C
Stacking gel 4% T, 3.8% C
Storage buffer Tris-acetate, pH 7.0
Shelf life 8 months at 2–8°C; expiration
date is printed on each cassette
Gel Percentage Optimum Separation range
7% 36–200 kD
3–8% 40–400 kD
Migration charts for protein standards
on Criterion XT Tris-Acetate gels.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
11
Criterion Precast Gels
3.3 SDS-PAGE Buffers
Table 3.2. Recommended Criterion precast gels and buffers for SDS-PAGE.
Gel Type
Sample Buffer
Running Buffer
Criterion TGX
Criterion TGX Stain-Free
Criterion Tris-HCl
Criterion Stain Free
Laemmli (catalog #161-0737)
Optional: 2-mercaptoethanol
(catalog #161-0710) or DTT
(catalog #161-0611)
Tris/glycine/SDS (catalog #161-0732)
Criterion XT Bis-Tris
XT sample buffer (catalog #161-0791)
Optional: XT reducing agent
(catalog #161-0792)
XT MES (catalog #161-0789)
XT MOPS (catalog #161-0788)
Criterion XT Tris-acetate
XT Tricine (catalog #161-0790)
3.3.1 Running Buffers
See Appendix B for buffer formulations. Do not adjust pH.
Tris/glycine/SDS (1x) (pH 8.3)
25 mM Tris, 192 mM glycine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH2O
XT MES (pH 6.4)
Dilute 50 ml 20x stock (catalog #161-0789) with 950 ml diH2O
XT MOPS (pH 6.9)
Dilute 50 ml 20x stock (catalog #161-0788) with 950 ml diH2O
XT Tricine (pH 8.2)
Dilute 50 ml 20x stock (catalog #161-0790) with 950 ml diH2O
3.3.2 Sample Buffers
Laemmli
62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% (catalog #161-0737)
bromophenol blue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)
XT
Use XT sample buffer (catalog #161-0791) and XT reducing agent
(catalog #161-0792)
3.4 Sample Preparation
1. Determine the appropriate concentration of sample to load (depends on the load volume and the
detection method used; see Chapter 12 for approximate stain sensitivities).
2. Dilute the sample with at least an equivalent volume of sample buffer with added reducing agent.
For nonreducing conditions, omit the reducing agent.
TGX and Tris-HCl Gels
XT Gels (Bis-Tris and Tris-Acetate)
4.75 μl Laemmli sample buffer 5 μl XT sample buffer
0.25 μl β-mercaptoethanol 1 μl XT reducing agent
5 μl sample
1–14 μl sample
10 μl total volume
Make up to 20 μl total volume with diH2O
3. Heat the diluted sample at 90–95°C for 5 min, or at 70°C for 10 min.
12
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
3.5 Running Conditions
Run conditions and times are approximate. Conditions may vary depending on water and buffer
conductivity, which vary from one lab setting to the next. Multiply current by the number of gels run.
Table 3.3. Running conditions for SDS-PAGE with Criterion gels in the Criterion cell. Do not run different gel
formulations at the same time.
TGXTris-HCl
Bis-Tris
Tris-Acetate
Running buffer
Tris/glycine/SDS
Tris/glycine/SDS
XT MOPS
XT MES
XT Tricine
Standard Conditions
Power conditions
200 V constant
200 V constant
200 V constant
200 V constant 150 V constant
55–80 mA
90–120 mA
165–175 mA
185–200 mA
170–180 mA
33–43 mA
35–55 mA
60–70 mA 90–110 mA
85–95 mA
42–45 min
50–55 min
60 min
45 min
65 min
—
—
—
—
Expected current (per gel)
Initial Final
Run time
High Voltage (Rapid) Conditions
Power conditions
300 V constant
Expected current (per gel)
Initial 89–135 mA
—
—
—
—
Final
66–99 mA
—
—
—
—
20–26 min
—
—
—
—
Run time
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
13
4
Native PAGE
4.1 Introduction
In native PAGE, proteins are prepared in nonreducing, nondenaturing sample buffer, which maintains
native structure and mass-to-charge ratios. Separation is also performed in the absence of SDS and
reducing agents. Though native PAGE uses the same moving boundary described for SDS-PAGE
(see Section 3.1), protein mobility depends on a number of factors besides molecular weight, including
the shape and charge of the protein. Protein-protein interactions may be retained during native PAGE,
so some proteins may separate as multisubunit complexes. Consequently, native PAGE is not suitable
for molecular weight determination.
The nonreducing and nondenaturing environment of native PAGE allows protein separation with
retention of biological activity. Because native structure is retained, native PAGE can allow resolution of
proteins with the same molecular weight.
4.2 Criterion™ Gel Selection Guide for Native PAGE
Table 4.1. Criterion precast gels for SDS-PAGE.
Gel Formulation
Gels
Laemmli-likeCriterion™
Description
TGX™
Laemmli-like, extended shelf life gels
Criterion™ TGX Stain-Free™
Laemmli-like, extended shelf life gels with trihalo compounds for rapid fluorescence detection
Tris-HCl (Laemmli)
Tris-HCl Laemmli gels
Criterion Tris-HCl
Criterion Stain Free™
Tris-HCl Laemmli gels with trihalo compounds for rapid fluorescence detection
Tris-acetateCriterion™ XT Tris-acetate
Based on a Tris-acetate buffer system (pH 7.0)
4.2.1 Criterion TGX and Criterion TGX Stain-Free Gels
Criterion TGX (Tris-Glycine eXtended shelf life) gels are Tris-HCl, Laemmli-like gels with a proprietary
modification that extends their shelf life to 12 months and enhances separation characteristics relative to
conventional gel types. The TGX formulation yields Laemmli-like separation patterns with exceptionally
straight lanes and sharp bands and has excellent staining quality and transfer efficiency.
These gels are run using native sample buffer and Tris/glycine running buffer.
Two types of TGX formulations are available:
Criterion TGX — Laemmli-like gels with the TGX formulation
n
Criterion TGX Stain-Free — Laemmli-like, extended shelf life gels that include unique trihalo
compounds that allow rapid fluorescent detection of proteins with the Criterion Stain Free
system, eliminating staining and destaining steps for faster results (see Chapter 5 for more
details)
n
14
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Gel Composition
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Shelf life ~12 months at 2–8°C; expiration date is printed on each cassette
4.2.2 Criterion Tris-HCl and Criterion Stain Free Gels
These Tris-HCl Laemmli gels are run using native sample buffer and Tris/glycine running buffer.
Criterion Tris-HCl — Tris-HCl, Laemmli-like formulation that offer a shelf life of 12 weeks
n
Criterion Stain Free — Tris-HCl, Laemmli-like gels with unique trihalo compounds that allow
rapid fluorescent detection of proteins with the Criterion Stain Free system, eliminating
staining and destaining steps for faster results (see Chapter 5 for more details)
n
Gel Composition
Gel buffer 0.375 M Tris-HCl, pH 8.6
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.6
Shelf life ~12 weeks at 2–8°C; expiration date is printed on each cassette
4.2.3 Criterion XT Tris-Acetate Gels
Criterion XT Tris-acetate gels can also be used to separate proteins by their charge-to-mass ratio
(under native PAGE conditions). Separation by native PAGE with XT Tris-acetate gels uses discontinuous
acetate and Tricine ion fronts to form moving boundaries to stack and separate proteins by both size
and charge.
Gel Composition
Gel buffer Tris-acetate, pH 7.0
Crosslinker 3.8% C
Stacking gel 4% T, 3.8% C
Storage buffer Tris-acetate, pH 7.0
Shelf life 8 months at 2–8°C; expiration
date is printed on each cassette
4.3 Native PAGE Buffers
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
Running buffer (1x) 25 mM Tris, 192 mM glycine
Dilute 100 ml 10x stock (catalog #161-0734) with 900 ml diH2O
Sample buffer 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 0.01% bromophenol blue
(catalog #161-0738)
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
15
Criterion Precast Gels
4.4 Sample Preparation
In the absence of SDS, the net charge of a polypeptide is determined by its amino acid composition and
the pH of the sample buffer. Only polypeptides with a net negative charge migrate into Criterion gels
under native conditions. Most polypeptides have an acidic or slightly basic pI (~3–8). These proteins can
be separated using the following standard protocol:
1. Determine the protein concentration and load volume of your sample based on the detection
method used (see Chapter 12 for approximate stain sensitivities).
2. Dilute the sample in twice the volume of native sample buffer (DO NOT HEAT SAMPLES).
For example, combine: 5 μl sample
10 μl native sample buffer (catalog #161-0738)
15 μl total volume
Strongly basic proteins (pl >8.5) have a net positive charge and will not enter a Criterion gel under native
conditions. To allow polypeptides with a net positive charge to migrate into a native Criterion gel, change
the polarity of the electrodes by reversing the color-coded jacks when connecting to the power supply.
4.5 Running Conditions
Running conditions for native PAGE are similar to the standard running conditions used for SDS-PAGE
(see Section 3.5). If high temperature is a concern, run native PAGE at lower voltage; at lower voltages, runs
require more time to complete.
Table 4.1. Running conditions for native PAGE for Criterion gels in the Criterion cell.
Running buffer
Power conditions
Laemmli/Laemmli-likeTris-Acetate
Native
Native
200 V constant
200 V constant
Expected current (per gel)
Initial
90–120 mA
70–80 mA
Final
35–55 mA
25–35 mA
55 min
75 min
Run time
16
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Criterion Stain Free™
System
5
5.1 Introduction
The Criterion Stain Free system, which comprises the Gel Doc™ EZ imager, Image Lab™ software,
and Criterion™ TGX Stain-Free™ and Criterion Stain Free precast gels, eliminates the time-consuming
staining and destaining steps required by other protein detection methods. Criterion TGX (Tris-Glycine
eXtended shelf life) Stain-Free gels include a proprietary modification that extends their shelf life to
12 months and enhances separation characteristics relative to conventional gel types. Criterion TGX
Stain-Free and Criterion Stain Free gels also include unique trihalo compounds that allow rapid
fluorescent detection of proteins with the Gel Doc EZ imager — without staining.
The trihalo compounds in the gels react with tryptophan residues in a UV-induced reaction to
produce fluorescence, which can be easily detected by the Gel Doc EZ imager within gels or on lowfluorescence PVDF membranes. Activation of the trihalo compounds in the gels adds 58 Da moieties
to available tryptophan residues and is required for protein visualization. Proteins that do not contain
tryptophan residues cannot be detected using this system. The sensitivity of the Criterion Stain Free
system is comparable to staining with Coomassie Brilliant Blue for proteins with a tryptophan content
>1.5%; sensitivity superior to Coomassie staining is possible for proteins with a tryptophan content >3%.
Molecular weights of proteins are estimated by a regression method using Image Lab software. The
software generates a standard curve using the molecular weight and relative mobility (Rf ) of standard
proteins (Rf = distance migrated by the protein/distance migrated by the dye front). The standard curve
is then used to estimate the molecular weights of sample proteins.
Benefits of the Criterion Stain Free system include:
Elimination of staining and destaining steps for faster results
n
Automated gel imaging and analysis
n
No background variability within a gel or between gels (as is often seen with standard
Coomassie staining)
n
Reduced organic waste by not requiring acetic acid and methanol for staining or destaining
n
Visualization of transferred (blotted) proteins on low-fluorescence PVDF membranes
n
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
17
Criterion Precast Gels
5.2 Criterion Stain Free Workflow
Perform Electrophoresis
SDS-PAGE (Chapter 3)
Native PAGE (Chapter 4)
2-D Electrophoresis (Chapter 11)
Activate/Image Gels
(Chapter 5)
Stain the Gels for Total Protein
Blot the Gels
(Chapter 12)
(Chapter 13)
Analyze the Separation
5.3 Electrophoresis with Criterion TGX Stain-Free Gels
Criterion TGX Stain-Free gels (and Criterion Stain Free gels) are made and packaged without SDS, so
they can be used for both SDS and native PAGE applications. To perform electrophoresis with these
gels, prepare the sample and running buffers, set up the Criterion cell, and perform the run as directed
in Chapters 2–4.
Use unstained standards with Criterion TGX Stain-Free and Criterion Stain Free gels,
as some prestained standards are not detected by Stain-Free technology.
To monitor electrophoresis, use 10 µl of a 1:1 mixture of Precision Plus Protein™ unstained
and Precision Plus Protein All Blue protein standards.
5.4 Using the Gel Doc EZ Imager
Image Criterion TGX Stain-Free and Criterion Stain Free gels and blots in the Gel Doc EZ imager.
The imager activates the reaction between the proteins and trihalo compounds in the gel to enable
visualization.
Immediately place the gel in the tray of the imager; no fixation or rinsing steps are required.
Prolonged rinsing may diminish image quality and lead to gel deformation
n
If desired, stain the gel with any TGX-compatible stains after imaging. Certain stains, if used
prior to imaging, eliminate detection capability
n
Refer to the Gel Doc EZ Stain-Free Sample Tray Instruction Manual (bulletin 10019634) for detailed
instructions.
18
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
6
Peptide Analysis
6.1 Introduction
Criterion™ Tris-Tricine/peptide gels are optimized for separating peptides and proteins with molecular
weight <10,000. Peptide-SDS complexes move more slowly through these gels, allowing the faster SDS
micelles that normally interfere with peptide separations to separate completely from peptides. This
enables resolution of distinct peptide bands.
6.2 Criterion Tris-Tricine/Peptide Gels
6.2.1 Gel Composition
Gel buffer 1.0 M Tris-HCl, pH 8.45
Crosslinker 2.6% C
Stacking gel
4% T, 2.6% C
Storage buffer 1.0 M Tris-HCl, pH 8.45
Shelf life ~12 weeks at 2–8°C; expiration date is printed on each cassette
6.2.2 Gel Selection Guide
Criterion Tris-Tricine/peptide gels are available in either a single percentage or a linear gradient format.
Gel Percentage
16.5% Optimum Separation Range
1.5–30 kD
10–20% 1–40 kD
6.3 Tris-Tricine/Peptide Buffers
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
Running buffer (1x) 100 mM Tris, 100 mM Tricine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0744) with 900 ml diH2O
Sample buffer
(catalog #161-0739)
200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie
Brilliant Blue G-250, 2% β-mercaptoethanol or 100 mM DTT (added fresh)
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
19
Criterion Precast Gels
6.4 Sample Preparation
1. Determine the appropriate concentration of sample to load (depends on the load volume and the
detection method used; see Chapter 12 for approximate stain sensitivities).
2. Dilute the sample with at least an equivalent volume of sample buffer (catalog #161-0739) and
reducing agent (β-mercaptoethanol, for example). Heat the diluted sample at 90–95°C for 5 min,
or at 70°C for 10 min.
For example, combine: 5 μl sample
4.75 μl Tricine sample buffer (catalog #161-0739)
0.25 μl β-mercaptoethanol (catalog #161-0710)
10 μl total volume
6.5 Running Conditions
20
Power conditions 125 V constant
Starting current 140–150 mA/gel
Final current 60–70 mA/gel
Run time 120 min
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Isoelectric Focusing
(IEF)
7
7.1 Introduction
Isoelectric focusing (IEF) separates proteins by their net charge rather than molecular weight. Criterion™
IEF gels are cast with Bio-Rad’s Bio-Lyte® ampholytes, amphoteric molecules that generate a pH
gradient across the gels. Proteins migrate to their isoelectric point (pI), the pH at which the protein
has no net charge. Criterion IEF gels contain no denaturing agents, so IEF is performed under native
conditions.
7.2 Criterion IEF Gels
7.2.1 Gel Composition
Gel buffer 2% ampholyte, pH 3–10 or 5–8
Crosslinker 3.0% C
Stacking gel None
Storage buffer diH2O
Shelf life ~24 weeks at 2–8°C; expiration date is printed on each cassette
7.2.2 Gel Selection Guide
IEF gel pH Range
5–8 5–8.0
3–10 4–8.5
7.3 IEF Buffers
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
Running buffers:
IEF cathode buffer (1x) 20 mM lysine (free base), 20 mM arginine (free base)
Dilute 100 ml 10x stock (catalog #161-0762) with 900 ml diH2O
IEF anode buffer (1x)
7 mM phosphoric acid
Dilute 100 ml 10x stock (catalog #161-0761) with 900 ml diH2O
Sample buffer 50% glycerol
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
21
Criterion Precast Gels
7.4 Sample Preparation
1. Determine the appropriate concentration of sample to load (depends on the load volume and the
detection method used).
2. Dilute the sample with at least an equivalent volume of sample buffer.
For example, combine: 5 μl sample
5 μl sample buffer
10 μl total volume
7.5 Running Conditions
22
Power conditions (stepwise)
100 V constant 60 min
250 V constant 60 min
500 V constant 30 min
Starting current 5–25 mA/gel
Final current
5–25 mA/gel
Run time 150 min
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
8
Protease Analysis by
Zymogram PAGE
8.1 Introduction
Criterion™ zymogram gels are used to test for proteolytic activity. Gels are cast with gelatin or casein,
which acts as a substrate for proteases that are separated in the gel under nonreducing conditions.
Proteases are detected by first renaturing the enzymes and then allowing them to break down the
substrate. Zymogram gels are stained with Coomassie Brilliant Blue R-250 stain, which stains the
substrate while leaving clear areas around active proteases.
8.2 Criterion Zymogram Gels
8.2.1 Gel Composition
Gel buffer 0.375 M Tris-HCl, pH 8.6
Crosslinker 2.6% C
Stacking gel 4% T, 2.6% C
Storage buffer 0.375 M Tris-HCl, pH 8.6, 0.2% NaN3
Shelf life ~8 weeks at 2–8°C; expiration date is printed on each cassette
8.2.2 Gel Selection Guide
Zymogram Gel Optimum Separation Range
10% with gelatin 30–150 kD
12.5% with casein 20–120 kD
8.3 Zymogram Buffers
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
Running buffer (1x)
25 mM Tris, 192 mM glycine, 0.1% SDS
Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH2O
Sample buffer
(catalog #161-0764)
62.5 mM Tris-HCl, pH 6.8, 4% SDS, 25% glycerol, 0.01% Coomassie
Brilliant Blue G-250
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
23
Criterion Precast Gels
8.4 Sample Preparation
1. Determine the appropriate protein concentration of your sample based on the detection method
and load volume used. (See Chapter 12 for approximate stain sensitivities.)
2. Dilute 1 part sample with 1 part sample buffer. Do not heat the samples.
8.5 Running Conditions
24
Power conditions 125 V constant
Starting current 90–120 mA/gel
Final current 35–55 mA/gel
Run time 90 min
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Nondenaturing Nucleic
Acid PAGE
9
9.1 Introduction
Criterion™ TBE gels are used to separate small double-stranded DNA (dsDNA) fragments, particularly
PCR products. DNA molecules have nearly uniform mass-to-charge ratios, allowing nondenaturing
nucleic acid PAGE to separate dsDNA by mass using a continuous TBE buffer system.
9.2 Criterion TBE Gels
9.2.1 Gel Composition
Gel buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3
Crosslinker 3.3% C
Stacking gel 4% T, 3.3% C
Storage buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA
Shelf life ~12 weeks at 2–8°C; expiration date is printed on each cassette
9.2.2 Gel Selection Guide
Gel Percentage
Optimum Separation Range
5%
200–2,000 bp
10%
50–1,500 bp
15%
20–1,000 bp
4–20%
10–2,000 bp
9.3 Nondenaturing Nucleic Acid PAGE Buffers
See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.
Running buffer (1x) 89 mM Tris, 89 mM boric acid, 2 mM EDTA
Dilute 100 ml 10x stock (catalog #161-0733) with 900 ml diH2O
Sample buffer (5x) (catalog #161-0767)
50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 25% glycerol, 0.2% bromophenol
blue, 0.2% xylene cyanole FF
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
25
Criterion Precast Gels
9.4 Sample Preparation
Determine the DNA concentration of your sample based on the detection method used. (See Chapter
12 for approximate stain sensitivities.) Dilute 4 parts sample with 1 part sample buffer.
9.5 Running Conditions
Table 9.1. Running conditions for nondenaturing nucleic acid PAGE with Criterion gels in the Criterion cell.
Power conditions
5% and 10% Gels
15% and 4–20% Gels
100 V constant
150 V constant
Expected current (per gel)
Initial
20–25 mA
27–35 mA
Final
14–18 mA
20–35 mA
90 min
90 min
Run time
26
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
10
Denaturing Nucleic
Acid PAGE
10.1 Introduction
Criterion™ TBE-urea gels are used for separation of small RNA and single-stranded DNA (ssDNA)
fragments. Applications include oligonucleotide analysis, RNase protection assays, and northern
blotting.
10.2 Criterion TBE-Urea Gels
10.2.1 Gel Composition
Gel buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.3
Crosslinker 3.3% C
Stacking gel 4% T, 3.3% C
Storage buffer 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3
Shelf life
~8 weeks at 2–8°C; expiration date is printed on each cassette
10.2.2 Gel Selection Guide
Gel Percentage
5%
Optimum Separation Range
50–1,000 nt
10%
25–300 nt
15%
10–50 nt
10.3 TBE-Urea Buffers
See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.
Running buffer (1x) 89 mM Tris, 89 mM boric acid, 2 mM EDTA
Dilute 100 ml 10x stock (catalog #161-0733) with 900 ml diH2O
Sample buffer (5x)
(catalog #161-0768)
89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0, 12% Ficoll,
0.01% bromophenol blue, 0.02% xylene cyanole FF, 7 M urea
10.4 Sample Preparation
Determine the desired ssDNA or RNA concentration for your sample based on the detection method
used. Dilute 4 parts sample with 1 part sample buffer.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
27
Criterion Precast Gels
10.5 Running Conditions
Table 10.1. Running conditions for denaturing nucleic acid PAGE with Criterion gels in the Criterion cell.
5% Gels
10% Gels
15% Gels
Power conditions
200 V constant
200 V constant
200 V constant
Expected current (per gel)
Initial
40–45 mA
20–33 mA
18–22 mA
Final
20–25 mA
14–18 mA
10–15 mA
90 min
90 min
90 min
Run time
28
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
11
2-D Electrophoresis
11.1 Introduction
Criterion™ precast gels are available for second-dimension PAGE in 2-D electrophoresis workflows.
The IPG-well gels accommodate 11 cm IPG strips. Criterion™ Any kD™ gels are particularly well suited
for 2-D electrophoresis applications.
The transition from first- to second-dimension gel electrophoresis involves:
Equilibration of the resolved IPG strips in a reducing buffer containing SDS
n
Placing the IPG strip on top of the second-dimension gel
n
11.2 Equilibration
Equilibration ensures that proteins in the IPG strips are coated with SDS and that cysteines are reduced
and alkylated. Use the equilibration protocols (bulletin 4110009) and buffers in the ReadyPrep™ 2-D
starter kit (catalog #163-2105), or other protocols and buffers used with Tris-HCl gels.
11.3 Agarose Overlay
Place the equilibrated IPG strip into the IPG well of the Criterion gel and overlay it with molten agarose to
ensure good contact between the strip and gel.
n
n
Criterion TGX™ and Tris-HCl gels: prepare 0.5% low-melt agarose (catalog #161-3111),
0.003% bromophenol blue (catalog #161-0404) in 1x Tris/glycine/SDS running buffer.
(Alternatively, use ReadyPrep overlay agarose, catalog #163-2111)
Criterion™ XT gels: prepare 0.5% low-melt agarose (catalog #161-3111), 0.001%
bromophenol blue (catalog #161-0404) in appropriate 1x XT running buffer
1. Following equilibration, place the IPG strip, gel side up, on the back plate of the Criterion gel, above
the IPG well. The “+” and pH range on the IPG strip should be on the left.
2. Using forceps, push the strip into the IPG well, taking care to not trap air bubbles under the strip.
Push on the backing of the strip, not on the gel.
3. Using a disposable pipet, add overlay agarose to the IPG well. Fill the well to the top of the inner
plate. Dispense rapidly, as overlay agarose solidifies quickly. To avoid bubbles, tilt the cassette
slightly to allow bubbles to escape. Push gently on the plastic backing of the strip to free any
trapped bubbles.
11.4 Second-Dimension Electrophoresis
Place the cassettes into the Criterion cell and start the run using the run conditions for SDS-PAGE.
Use the migration of the bromophenol blue in the overlay agarose to monitor the progress of the run.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
29
12
Detection
12.1 SDS-PAGE and Native PAGE Detection
Following electrophoresis, either stain the gel or use the Criterion Stain Free™ system to visualize
proteins in the gel.
Refer to Table 12.1 for a comparison of total protein stains
n
For Criterion™ TGX Stain-Free™ and Criterion Stain Free™ gels, immediately place the gel on
the tray of the Gel Doc™ EZ imager; no additional fixation or rinsing steps are required.
If desired, stain with any compatible stains (Table 12.1) following imaging. Some stains, if
used prior to imaging, can impair imaging quality
n
Table 12.1. Total protein gel stains for use with Criterion gels.
Sensitivity
(Lower
Limit)
Stain
Optimum
Protein Load
(µg/Band)
Advantages
Disadvantages
Imaging
Requires
methanol
destaining
Photography
with white light
or transmission
densitometry
Manual
Criterion Gels
Coomassie
R-250
36–47 ng
~0.5
Laboratory
standard
Bio-Safe™
Coomassie
8–28 ng
~0.5
Nonhazardous
Zinc stain1
6–12 ng
~0.2
High contrast, fast,
reversible
Negative SDSPAGE stain, must
be photographed
Silver
Stain Plus™ kit
0.6–1.2 ng
~0.01
Sensitive, robust,
mass spectrometry
compatible
Does not stain
glycoproteins
well
LIT442
Silver stain
0.6–1.2 ng
~0.01
Stains complex
proteins (glyco- or
lipoproteins)
Not mass
spectrometry
compatible
LIT34
Dodeca™
silver stain kit
0.5–1.2 ng
~0.1
Convenient
staining for a large
number of gels
~2 ng
~0.1
High sensitivity,
broad dynamic
range, simple
one-step protocol
Oriole™
fluorescent
gel stain1
1 Do
30
not use zinc stain or Oriole fluorescent gel stain to stain native gels.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Consult
literature
4307051
4006082
4110150
Fluorescence
visualization
with UV transillumination
10017295
Instruction Manual and Application Guide
Sensitivity
(Lower
Limit)
Stain
SYPRO
Ruby protein
gel stain
Flamingo™
fluorescent gel
stain
Optimum
Protein Load
(µg/Band)
1–10 ng
0.25–0.5 ng
Advantages
Disadvantages
Imaging
~0.1
Broad dynamic
range
~0.02
Broad dynamic
range, mass
spectrometry
compatible
Requires laseror LED-based
imaging
instrument for
maximum
sensitivity
Fluorescence
visualization
with UV,
LED, or laser
scanning
Manual
Requires
tryptophan
residues in
proteins for
detection
Fluorescence
visualization
using Criterion
Stain Free
imaging system
4006173
10003321
Criterion TGX Stain-Free and Criterion Stain Free Gels
Stain Free
imaging
2–28 ng
~0.5
Rapid (<5 min),
compatible with
blotting and mass
spectrometry,
simple protocol
with no additional
reagents
10014472
12.2 Peptide Gel Staining
Peptides and small proteins are prone to diffusion and loss during staining. The following protocol
includes a fixing step prior to staining to prevent sample loss and is suitable for detection of bands as
low as 10–20 ng.
Fixative solution
40% methanol, 10% acetic acid
Stain solution
0.025% (w/v) Coomassie Blue G-250, 10% acetic acid
Destain solution
10% acetic acid
Place gels in fixative solution and equilibrate for 30 min. Stain gels with stain solution for 1 hr. Stain
should be used only once; reuse may result in loss of sensitivity. Destain gels three times for 15 min or
until the desired background is achieved. Some peptides may not be completely fixed and may diffuse
out of the gels if fixing and staining times are greatly exceeded.
12.3 IEF Gel Staining
Samples on IEF gels can be detected using multiple methods. Use Table 12.2 as a guide to selecting an
appropriate staining method.
Table 12.2. IEF gel detection methods.
Sensitivity
(Lower Limit)
Optimum
Protein Load
(µg/Band)
Silver Stain Plus kit
0.6–1.2 ng
~0.01
Sensitive, robust, mass
spectrometry compatible
Requires TCA fixation
Silver stain
0.6–1.2 ng
~0.01
Stains complex proteins
(glyco- or lipoproteins)
Not mass spectrometry
compatible
SYPRO Ruby protein
gel stain
1.0–10 ng
~0.1
Broad dynamic range, mass
spectrometry compatible
Requires laser- or LEDbased imaging instrument
for maximum sensitivity
Flamingo fluorescent
gel stain
0.25–0.5 ng
~0.2
Broad dynamic range, mass
spectrometry compatible
Requires laser- or LEDbased imaging instrument
for maximum sensitivity
Method
Advantages
Disadvantages
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
31
Criterion Precast Gels
12.4 Zymogram Gel Staining
Prior to staining zymogram gels, sample proteases must first be renatured and allowed to break down
the substrate contained in the gel. The following protocol provides basic guidelines for detection.
Optimal results should be determined empirically.
Renaturing solution
2.5% Triton X-100
Development solution
50 mM Tris, 200 mM NaCl, 5 mM CaCl2 (anhydrous), 0.02% Brij-35
Adjust to pH 7.5
Staining solution
40% methanol, 10% acetic acid, 0.5% Coomassie Blue R-250
Destaining solution
40% methanol, 10% acetic acid
Place gels in renaturing solution for 30 min at room temperature. Incubate gels in development solution
at 37ºC for a minimum of 4 hr. Highest sensitivity is typically achieved with overnight incubation.
Optimum conditions should be determined empirically. Stain gels with staining solution for at least 1 hr
at room temperature. Destain until clear bands appear against the blue background (~30–60 min).
12.5 TBE Gel Staining
Use Table 12.3 as a guide to selecting an appropriate staining method.
Table 12.3. TBE gel detection methods.
Method
Ethidium bromide
Silver stain
SYBR® Green
®
SYBR Safe
Sensitivity
(Lower Limit)
50 ng
1–2 ng
Advantages
Disadvantages
Classic fluorescent DNA stain
Carcinogenic
More sensitive than ethidium bromide
Requires multiple steps
0.02–2 ng
High sensitivity
Multiple steps, –20°C storage
0.5 ng
Non-hazardous
Multiple steps
12.6 TBE-Urea Gel Staining
Use Table 12.4 as a guide to selecting an appropriate staining method.
Table 12.4. TBE-urea gel detection methods.
Method
Advantages
Disadvantages
Ethidium bromide
10 ng
Classic fluorescent DNA stain
Carcinogenic
Radiant™ Red
10 ng
Fast, single-step protocol
RNA and ssDNA only
More sensitive than ethidium bromide
Requires multiple steps
Silver stain
32
Sensitivity
(Lower Limit)
1–2 ng
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
13
Blotting
13.1 Introduction
Western blotting is an electrophoretic technique used to move proteins from a gel onto a solid support
such as a nitrocellulose or PVDF membrane. The membrane can be used for immunological or
biochemical analyses or demonstration of protein-protein or protein-ligand interactions. Below are
guidelines for western blotting of Criterion™ precast gels onto nitrocellulose or PVDF membranes using
either wet or semi-dry transfer techniques.
Assess transfer efficiency using a total protein blot stain such as SYPRO Ruby stain (see Table 12.1).
With Criterion™ TGX Stain-Free™ and Criterion Stain Free™ gels, transfer efficiency to low-fluorescence
PVDF membranes may also be assessed using the Gel Doc™ EZ imager (see Chapter 5; activate the gel
before blotting).
13.2 Transfer
13.2.1 Transfer Buffers
See Appendix B for buffer formulations. Do not adjust pH unless directed to do so.
Towbin buffer (1x) 25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)
Dilute 100 ml 10x stock (catalog #161-0734) with 400 ml diH2O.
Add 200 ml methanol, then adjust volume to 1 L with diH2O.
Add SDS to 0.1% to promote transfer of high molecular weight proteins.
13.2.2 Wet Transfer Using the Criterion Blotter
1. Equilibrate the gels and membranes (for example, in Towbin buffer) for 15 min prior to blot assembly.
2. Assemble the transfer apparatus:
a) Fill the tank with transfer buffer to ~50% of the fill volume and place a magnetic stir bar inside
the tank.
b) Place the ice block in the pocket in the back of the cell. Flip down the lever to hold the ice
block. Alternatively, connect the optional cooling coil to an appropriate recirculating water chiller
and place it in the grooves in the back of the tank.
3. Assemble the cassette:
a) Pour chilled transfer buffer into each compartment of the assembly tray, and then place the
membrane (nitrocellulose, PVDF) into the front (small) compartment of the tray to soak.
Wet PVDF membranes in methanol before soaking in transfer buffer.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
33
Criterion Precast Gels
b) Open the cassette and place it in the back (large) compartment of the tray so the red plate
with the handle is vertical (anode) and the black plate (cathode) is horizontal and submerged in
transfer buffer.
c) Assemble the sandwich as shown, placing the gel closest to the black side of the cassette and
the membrane closest to the red plate. Use a blot roller to remove air trapped between the
layers of the blot assembly.
Foam pad
Filter paper
Membrane
Gel
Filter paper
Foam pad
Assembly of the gel blot sandwich with the Criterion blotter.
4. Place the assembled cassette into the groove in the tank, aligning the red side of the card with the
red electrode. Make sure the magnetic stirbar is free to move. Repeat steps 2–4 for a second blot.
5. Add the remaining transfer buffer to the fill level marked on the tank, place the tank on a stir plate,
and begin stirring to maintain even buffer temperature and ion concentration during the transfer.
6. Connect the Criterion blotter to a PowerPac™ HC power supply and begin transfer.
For many proteins, excellent transfer efficiency is obtained in 30 min at a constant voltage of 100 V. For
best results, optimize conditions for proteins of interest. Large proteins (>150 kD) may take 60 min, while
smaller proteins (<30 kD) may transfer in 20 min. Refer to the Criterion Blotter Instruction Manual (bulletin
4006190) or the Protein Blotting Guide (bulletin 2895) for additional information.
13.2.3 Transfer Using the Trans-Blot® Turbo™ System
1. Open the transfer pack and assemble the components on the cassette in the order shown. For best
results, use the roller to remove any air trapped between the layers. If using a single mini or midi
sandwich, place the sandwich in the middle of the cassette bottom. With two mini gels, place the
sandwiches on a midi stack with the foot of each gel facing the center of the stack.
2. Place the lid on the cassette and lock the lid in place by turning the knob clockwise, using the
symbols on the lid as a guide. Slide the cassette into the appropriate bay of the Trans-Blot Turbo cell.
Each cassette and bay can hold up to two mini gels or one midi gel (Table 13.1).
3. Start the transfer. With the cassette in the cell, press TURBO and select the gel type. Press A:RUN
or B:RUN to begin the transfer. Press LIST to select a Bio-Rad optimized protocol (Table 13.2) or a
user-defined protocol. Press NEW to create and run a new protocol.
4. When transfer completes, RUN COMPLETE appears. Pull the cassette straight out of the slot and
unlock the lid. Disassemble the blotting sandwich.
34
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Cassette top
(–) electrode (cathode)
Top ion reservoir stack
Gel
Membrane
Bottom ion reservoir
stack
Cassette bottom
(+) electrode (anode)
Assembly of the gel blot sandwich with the Trans-Blot Turbo system.
Table 13.1. Placement of cassettes in the Trans-Blot Turbo cell.
Acceptable
Unacceptable
Option 1
Option 2
Option 1
Option 2
Upper bay (A)
1 mini gel
2 mini gels -or- 1 midi gel
1 mini gel
2 mini gels -or- 1 midi gel
-and/or-
-and/or-
-and-
-and-
Lower bay (B)
1 mini gel
2 mini gels -or- 1 midi gel
2 mini gels -or- 1 midi gel
1 mini gel
Table 13.2. Trans-Blot Turbo transfer protocols.
MW, kD
Time, Min
Protocol Name
STANDARD SD
1.5 MM GEL
HIGH MW
LOW MW
1 Mini Gel
Any
30
Up to 1.0 A, 25 V constant
2 Mini Gels or 1 Midi Gel
Up to 1.0 A, 25 V constant
Any
10
2.5 A constant, up to 25 V
1.3 A constant, up to 25 V
>150
10
2.5 A constant, up to 25 V
1.3 A constant, up to 25 V
<30
5
2.5 A constant, up to 25 V
1.3 A constant, up to 25 V
MIXED MW
5–150
7
2.5 A constant, up to 25 V
1.3 A constant, up to 25 V
1 Mini TGX
5–150
3
2.5 A constant, up to 25 V
N/A
Refer to the Trans-Blot Turbo Instruction Manual (bulletin 10020688) for complete instructions or the
Protein Blotting Guide (bulletin 2895) for additional information.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
35
Criterion Precast Gels
13.2.4 Semi-Dry Transfer Using the Trans-Blot® SD Cell
1. Equilibrate the gels and membranes (for example, in transfer buffer; see Appendix B for buffer
recipes) for 20 min prior to blot assembly.
2. Assemble the blot for transfer using the Trans-Blot SD semi-dry transfer system.
3. Connect the Trans-Blot SD cell to a PowerPac HC
power supply and begin transfer at 10–15 V.
For most proteins transferred from Criterion precast
gels, optimum transfer efficiency is obtained in
30 min; smaller proteins (<30 kD) may transfer more
quickly, while proteins >150 kD may show increased
transfer efficiencies at up to 60 min. Run times longer
than 60 min are NOT recommended for semi-dry
transfers.
(–)
Filter paper
Refer to the Trans-Blot SD Instruction Manual (bulletin
1703940) or the Protein Blotting Guide (bulletin 2895)
for additional information.
Gel
Membrane
Filter paper
13.3 Total Protein Blot Stains
Total protein staining of a membrane provides
an image of the complete protein pattern, which
is required for the full characterization of specific
antigens detected in complex protein mixtures.
Gels shrink during staining, so comparison of an
immunologically probed membrane to a stained gel is
not practical. Instead, the exact location of a specifc
antigen is determined by comparing two blotted
membranes: one that has been probed with an
antibody and the other stained for total protein.
(+)
Assembly of the gel blot sandwich with the
Trans-Blot SD cell.
Table 13.1. Total protein blot stains.
Method
SYPRO Ruby
protein blot stain
36
Sensitivity
Protein
Load
(μg/Band)
Advantages
Disadvantages
Imaging
2–8 ng
~0.2
Compatible with
mass spectrometry,
Edman-based
sequencing,
and standard
immunological
procedures
Multi-step protocol;
requires UV, LED,
or laser imaging for
maximum sensitivity
Fluorescence
visualization
with UV, LED
epi-illumination or
laser scanning
Colloidal gold stain
1 ng
~0.1
Highly sensitive,
single-step protocol
Incompatible with nylon
membranes
Anionic dyes
(amido black,
Coomassie R-250,
Ponceau S, Fast
Green FCF)
100–1,000 ng
~5.0
Inexpensive, rapid
Low sensitivity
Photography with
epi-illumination
or reflectance
densitometry
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
To visualize total protein on blots using the Gel Doc EZ imager, refer to Section 5.4.
13.4 Immunodetection
After transfer, blots are ready for downstream processing. While all protein and antibody combinations
are different and may require optimization, a general protocol for the immunodetection of a large
number of protein and antibody combinations is provided (see Appendix B for buffer formulations).
1. Immediately after transfer, place the membrane into Tris-buffered saline with Tween 20 (TTBS)
containing blocking agent (for example, 3% BSA, 5% nonfat dry milk, 1% casein, or 1% gelatin).
Incubate at room temperature with agitation for 1 hr.
2. Dilute the primary antibody in blocking solution (suggested dilution is specified by the manufacturer).
Incubate the blot in the primary antibody solution at room temperature and with agitation for 1 hr.
3. Wash the blot with TTBS as directed in the instructions for the detection method to be used (for
example, 5 times, 5 min each at room temperature).
4. Dilute the secondary antibody into TTBS as specified by the manufacturer. Incubate the blot in the
secondary antibody solution at room temperature with agitation for 1 hr.
5. Wash the blot with TTBS for 5 min at room temperature with agitation. Pour off the wash solution
and repeat 5 times.
6. Follow the directions for the detection kit used to develop the blot. For the Immun-Star™ WesternC™
chemiluminescence kit (catalog #170-5070), mix 3 ml luminol/enhancer with 3 ml peroxide solution
to make a 1x working solution for a 7 x 8.5 cm membrane. Incubate the membrane in the solution
for 3–5 min. Prior to imaging, drain the excess substrate and place the membrane in a protective
sleeve (such as plastic wrap) to prevent drying.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
37
14
Troubleshooting
Table 14.1. Troubleshooting electrophoresis and detection with Criterion™ gels. For more troubleshooting tips, refer to the
Criterion cell, Criterion blotter, and Trans-Blot® SD cell instruction manuals, or contact Technical Support.
Problem
Cause
Solution
General Troubleshooting Tips
Current is zero or less than
expected, and samples do not
migrate into gel
Gels run faster than expected
Bands “smile” across gel: band
pattern curves upward at both
sides of the gel
Bands “smile” or “frown” within gel
lanes
Bands are skewed or distorted;
lateral band spreading
Tape at bottom of cassette not removed
Remove tape
Insufficient buffer in integral buffer
chamber
Fill buffer chamber with running buffer
Insufficient buffer in lower buffer
chamber
Fill both halves of lower buffer tank with
400 ml running buffer when running two
gels
Electrical disconnection
Check electrodes and connections
Running buffer too concentrated or
incorrect
Check buffer composition
Gel temperature too high
Do not exceed recommended running
conditions
Excessive heating of gel
Check buffer composition
Do not exceed recommended running
conditions
Insufficient buffer
Fill both halves of lower buffer tank with
400 ml running buffer when running two
gels
Protein load too high
Load less protein
Sample or buffer preparation issues
Minimize salts, detergents, and solvents
in sample preparation and sample loading
buffers
Incorrect running conditions
Set correct voltage
Too much salt in samples
Remove salt from samples (dialysis,
precipitation, or other method)
Insufficient or wrong sample buffer
Check buffer composition and dilution
instructions
Sample precipitation
Selectively remove predominant proteins in
sample
Dilute sample in sample buffer
Insoluble materials (for example, cell
membranes) in samples
38
Centrifuge samples to remove particulates
prior to sample loading
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Problem
Artifactual bands at
60–70 kD
Cause
Skin keratin contamination
Solution
Clean all dishware, and wear gloves while
handling and loading gels
Filter all solutions (0.2–0.45 µm filter)
Poor resolution or fuzzy bands
Sample volume too high
If possible, load a more concentrated
sample in a lower sample buffer volume
Not enough running buffer in lower
chamber
Add running buffer to fill line of lower buffer
chamber
Diffuse sample loading zone
Load sample with a syringe or gel loading
pipet tip
Sample diffusion during staining
Fix gel with 40% methanol, 10% acetic acid
for 80 min prior to staining
Incompatible sample components
Minimize salts, detergents, and solvents
in sample preparation and sample loading
buffers
Expired gel
Use gels before expiration date printed on
the cassette
Criterion™ TGX Stain-Free™ and Criterion Stain Free™ Gels
Low sensitivity for proteins
Low tryptophan content in proteins
After activation and imaging, stain gel with
Bio-Safe™ Coomassie or similar to detect
missing bands
Uneven sensitivity or fuzzy bands
Gel was soaked in water or buffer prior
to activation and imaging
If possible, activate and image gel using
the Gel Doc EZ imager immediately after
electrophoresis
Bands are too light or missing from
blot (membrane)
Proteins transferred through membrane
Use membrane with smaller pore size
Decrease transfer time
Decrease voltage
Standards not visible
Incorrect standards were used
Use unstained standards; some prestained
standards are not detected by the Gel Doc
EZ imager. To monitor electrophoresis, use
a 1:1 mixture of unstained and prestained
standards
Dye front at bottom of gels
interfering with detection of proteins
Sample constituents present in gel
interfering with imaging
Dilute sample in gel running buffer prior to
loading
Activate and image gel, rinse in fixation
solution for 30 min, and repeat imaging
High background or low sensitivity
on a blot of a Criterion TGX StainFree or Criterion Stain Free gel
(imaged using the Gel Doc EZ
imager)
Low tryptophan content in proteins
After activation and imaging, stain blot with
a total protein blot stain to detect missing
bands
Membrane not low-fluorescence PVDF
Always use low-fluorescence PVDF
membranes to blot Criterion TGX Stain-Free
or Criterion Stain Free gels
PVDF membrane is dry
Wet PVDF membrane briefly in methanol
and wash in water for 1 min before imaging
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
39
A
Quick Start Guide
The following instructions are for electrophoresis of Criterion™ precast gels using the Criterion system.
Prepare Buffers
Running buffer (1x)
Dilute 100 ml 10x stock (catalog #161-0732) with 900 ml diH2O.
Sample buffer Use Laemmli sample buffer (catalog #161-0737).
Prepare Gels and
Assemble Electrophoresis Cell
Remove the comb and tape from the gels and assemble the electrophoresis cell.
Fill the inner and outer buffer chambers with running buffer.
Fill the lower buffer chamber with 400 ml running buffer (to the fill mark).
Prepare and Load Samples
Component
Reducing
Nonreducing
5 μl
5 μl
Laemmli sample buffer
(catalog #161-0737)
4.75 μl
5 μl
β-Mercaptoethanol
0.25 μl
—
10 μl
10 μl
Sample
Total volume
Heat samples at 90–100°C for 5 min or at 70°C for 10 min.
Load the appropriate amount of sample on the gel.
Perform Electrophoresis
Connect the electrophoresis cell to the power supply and perform electrophoresis according to
the conditions in Table A.1.
40
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Table A.1. Running conditions for SDS-PAGE in the Criterion cell. Do not run different gel formulations at the same time.
TGXTris-HCl
Bis-Tris
Tris-Acetate
Running buffer
Tris/glycine/SDS
Tris/glycine/SDS
XT MOPS
XT MES
XT Tricine
Standard Conditions
Power conditions
200 V constant
200 V constant
200 V constant
200 V constant 150 V constant
Expected current (per gel)
Initial 55–80 mA
90–120 mA
165–175 mA
185–200 mA
170–180 mA
Final
33–43 mA
35–55 mA
60–70 mA 90–110 mA
85–95 mA
42–45 min
50–55 min
60 min
45 min
65 min
—
—
—
—
Run time
High Voltage (Rapid) Conditions
Power conditions
300 V constant
Expected current (per gel)
Initial 89–135 mA
—
—
—
—
Final
66–99 mA
—
—
—
—
20–26 min
—
—
—
—
Run time
Table A.2. Standard transfer (blotting) conditions.
Method
MembraneTime Voltage
Tank blotting
Nitrocellulose (0.45 μm)
PVDF (0.2 μm)
20–60 min
100 V1
Semi-dry blotting
Nitrocellulose (0.45 μm)
20–60 min
25 V
1
PVDF (0.2 μm)
For previously optimized protocols for proteins <30 kD, reduce transfer time by 20%.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
41
B
Buffers
Running Buffers
42
10x SDS-PAGE (1 L)
(catalog #161-0732)
250 mM Tris, 1.92 M glycine, 1% SDS, pH 8.3
10x Native PAGE (1 L)
(catalog #161-0734)
250 mM Tris, 1.92 M glycine, pH 8.3
10x Tris-Tricine (1 L) (catalog #161-0744)
1 M Tris, 1 M Tricine, 1% SDS, pH 8.3
10x TBE (1 L)
(catalog #161-0741)
890 mM Tris, 890 mM boric acid, 20 mM EDTA
Tris base
Glycine
SDS
diH2O
Do not adjust the pH (~pH 8.3)
Tris base
Glycine
diH2O
Do not adjust the pH (~pH 8.3)
Tris base
Tricine
SDS
diH2O
Do not adjust the pH (~pH 8.3)
Tris base
Boric acid
EDTA
diH2O
Do not adjust the pH (~pH 8.3)
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
30.3 g
144.1 g
10 g
to 1 L
30.3 g
144.1 g
to 1 L
121.1 g
179.2 g
10 g
to 1 L
107.8 g
55.0 g
5.8 g
to 1 L
Instruction Manual and Application Guide
Sample Buffers
2x SDS-PAGE (Laemmli, 30 ml) 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% (catalog #161-0737)
bromophenol blue, 5% β-mercaptoethanol (added fresh)
0.5 M Tris-HCl, pH 6.8
50% Glycerol
1.0% Bromophenol blue
10% SDS
diH2O
3.75 ml
15.0 ml
0.3 ml
6.0 ml
to 30 ml
Add β-mercaptoethanol (50 µl to 950 µl sample buffer)
before use.
2x Native PAGE (30 ml) 62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% (catalog #161-0738)bromophenol blue
0.5 M Tris-HCl, pH 6.8
50% Glycerol
1.0% Bromophenol blue
diH2O
2x Tricine (30 ml) (catalog #161-0739)
200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie Brilliant Blue G-250, 2% β-mercaptoethanol (added fresh)
1.0 M Tris-HCl, pH 6.8
100% Glycerol
10% SDS
Coomassie Blue G-250
diH2O
3.75 ml
24 ml
0.3 ml
to 30 ml
6.0 ml
12.0 ml
6.0 ml
12.0 mg
to 30 ml
Add β-mercaptoethanol (20 µl to 980 µl sample buffer)
before use.
5x Nucleic acid (10 ml)
(catalog #161-0767)
50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 25% glycerol, 0.2% bromophenol blue, 0.2% xylene cyanole FF
Tris base
50% Glycerol
EDTA
1.0% Bromophenol blue
Xylene cyanole FF
diH2O
78.8 mg
5 ml
14.6 mg
2.0 ml
20.0 mg
to 10 ml
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
43
Criterion Precast Gels
TBE-urea (30 ml)
(catalog #161-0768)
Store at 4°C 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0, 12% Ficoll, 0.01% bromophenol blue, 0.02% xylene cyanole,
7 M urea
Tris base
Boric acid
EDTA
Ficoll
Bromophenol blue
Xylene cyanole FF
Urea
diH2O
0.32 g
0.165 g
17.5 mg
3.6 g
3 mg
6 mg
12.6 g
to 30 ml
Buffer Components
0.5 M Tris-HCl, pH 6.8 (1 L)
(catalog #161-0799)
Store at 4°C Tris base
diH2O
Adjust to pH 6.8 with HCl
diH2O 10% SDS (250 ml)
(catalog #161-0416)
SDS
diH2O
1.0% Bromophenol blue (10 ml)
(10 g powder, catalog #161-0404)
Bromophenol blue
diH2O 60.6 g
~900 ml
to 1 L
25.0 g
to 250 ml
100 mg
to 10 ml
Blotting Buffers
Towbin buffer (1 L)
25 mM Tris, 192 mM glycine, 20% methanol
Dissolve:
Tris base
Glycine
diH2O
14.4 g
3.03 g
500 ml
Then add:
Methanol
diH2O
200 ml
to 1 L
Alternatively, use:
10x Tris/glycine (catalog #161-0734)
100 ml
Add 200 ml methanol and diH2O to 1 L as above
Tris-buffered saline with
Tween (TTBS, 1 L)
20 mM Tris, 500 mM NaCl, 0.05% Tween 20
Tris base
NaCl
10% Tween 20
diH2O
2.4 g
29.2 g
5.0 ml
to 1 L
Alternatively, use:
10x TBS (catalog #170-6435)
100 ml
10% Tween 20 (catalog #166-2404)
5 ml
diH2O895 ml
44
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
C
Related Literature
Bulletin #
Title
4006183 Criterion™ Cell Instruction Manual
4006197 Criterion™ Dodeca™ Cell Instruction Manual
10014472 Gel Doc™ EZ System Installation Guide
10019634
Stain-Free Sample Tray Instruction Manual
4006190
Criterion Blotter Instruction Manual
4006066 Trans-Blot® SD Semi-Dry Transfer Cell Quick Reference Guide
10020688 Trans-Blot® Turbo™ Blotting System Instruction Manual
1703940 Trans-Blot SD Semi-Dry Transfer Cell Instruction Manual
6007Criterion™ TGX™ Precast Gels Product Information Sheet
5974Criterion™ TGX Stain-Free™ Precast Gels Product Information Sheet
2911 Criterion™ XT Precast Gels Product Information Sheet
2895
Protein Blotting Guide
6039
Trans-Blot Turbo Transfer System Brochure
3133
Molecular Weight Determination by SDS-PAGE
3144
Using Precision Plus Protein™ Standards to Determine Molecular Weight
1939
Blotting Membrane Brochure
2032
Western Blotting Detection Reagents Brochure
2317
Ready-to-Run Buffers and Solutions Brochure
2414
The Little Book of Standards
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
45
Criterion Precast Gels
D
Ordering Information
Criterion TGX™ Gels
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
IPG+1 Well
(11 cm IPG Strip)
Prep+2 Well
(800 µl/well)
7.5%
567-1023
567-1024
567-1025
567-1021
567-1022
10%
567-1033
567-1034
567-1035
567-1031
567-1032
12%
567-1043
567-1044
567-1045
567-1041
567-1042
18%
567-1073
567-1074
567-1075
567-1071
567-1072
4–15%
567-1083
567-1084
567-1085
567-1081
567-1082
4–20%
567-1093
567-1094
567-1095
567-1091
567-1092
8–16%
567-1103
567-1104
567-1105
567-1101
567-1102
10–20%
567-1113
567-1114
567-1115
567-1111
567-1112
™
567-1123
567-1124
567-1125
567-1121
567-1192
Any kD
Criterion™ TGX Stain-Free™ Gels
46
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
IPG+1 Well
(11 cm IPG Strip)
Prep+2 Well
(800 µl/well)
7.5%
567-8023
567-8024
567-8025
567-8021
567-8022
10%
567-8033
567-8034
567-8035
567-8031
567-8032
12%
567-8043
567-8044
567-8045
567-8041
567-8042
18%
567-8073
567-8074
567-8075
567-8071
567-8072
4–15%
567-8083
567-8084
567-8085
567-8081
567-8082
4–20%
567-8093
567-8094
567-8095
567-8091
567-8092
8–16%
567-8103
567-8104
567-8105
567-8101
567-8102
10–20%
567-8113
567-8114
567-8115
567-8111
567-8112
Any kD
567-8123
567-8124
567-8125
567-8121
567-8122
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Criterion™ Tris-HCl Gels
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
IPG+1 Well
(11 cm IPG Strip)
Prep+2 Well
(800 µl/well)
5%
345-0001
345-0002
345-0003
—
—
7.5%
345-0005
345-0006
345-0007
—
345-0008
10%
345-0009
345-0010
345-0011
345-0101
345-0012
12.5%
345-0014
345-0015
345-0016
345-0102
345-0017
15%
345-0019
345-0020
345-0021
—
345-0022
18%
345-0023
345-0024
345-0025
—
345-0026
Criterion Tris-HCl
4–15%
345-0027
345-0028
345-0029
345-0103
345-0030
4–20%
345-0032
345-0033
345-0034
345-0104
345-0035
8–16%
345-0037
345-0038
345-0039
345-0105
345-0040
10.5–14%
345-9949
345-9950
345-9951
345-0106
—
10–20%
345-0042
345-0043
345-0044
345-0107
345-0045
™
Criterion Stain Free
10%
345-1012
345-1018
—
—
—
4–20%
345-0412
345-0418
345-0426
—
—
8–16%
345-8162
—
345-8166
345-8161
—
Criterion™ XT Gels
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
IPG+1 Well
(11 cm IPG Strip)
Prep Well
(800 µl/well)
345-0112
345-0113
345-0115
—
Criterion XT Bis-Tris
10%
345-0111
12%
345-0117
345-0118
345-0119
345-0121
345-0120
4–12%
345-0123
345-0124
345-0125
345-0127
345-0126
Criterion XT Tris-Acetate
7%
345-0135
345-0136
345-0137
—
—
3–8%
345-0129
345-0130
345-0131
345-0133
—
Criterion Tris-Tricine Peptide Gels
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
Prep+2 Well
(800 µl/well)
16.5%
345-0063
345-0064
345-0065
345-0066
10–20%
345-0067
345-0068
345-0069
—
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
pH 3–10
345-0071
345-0072
345-0073
pH 5–8
—
345-0076
—
Criterion IEF Gels
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
47
Criterion Precast Gels
Criterion Zymogram Gels
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
10% Zymogram, gelatin
345-0079
345-0080
345-0081
12.5% Zymogram, casein
345-0082
345-0083
345-0084
Criterion TBE Gels
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
5% TBE
345-0047
345-0048
345-0049
10%
345-0051
345-0052
345-0053
15%
345-0055
345-0056
345-0057
4–20%
345-0059
345-0060
345-0061
12+2 Well
(45 µl/well)
18-Well
(30 µl/well)
26-Well
(15 µl/well)
—
345-0086
—
10%
345-0088
345-0089
345-0090
15%
345-0091
345-0092
345-0093
Criterion TBE-Urea Gels
5% TBE-urea
Catalog # Description
Criterion Gel Accessories
345-9901 Criterion Empty Cassettes, 1.0 mm with 12+2 well comb, 10
345-9902 Criterion Empty Cassettes, 1.0 mm with 18-well comb, 10
345-9903 Criterion Empty Cassettes, 1.0 mm with 26-well comb, 10
345-9904 Criterion Empty Cassettes, 1.0 mm with prep+2 well comb, 10
345-9906 Criterion Empty Cassettes, 1.0 mm with IPG+1 well comb, 10
165-6006
Criterion Sample Loading Guide, 12+2 well, 1
165-6007 Criterion Sample Loading Guide, 18-well, 1
165-6008 Criterion Sample Loading Guide, 26-well, 1
Equipment
165-6001 Criterion Cell, includes tank, lid with power cables, three sample loading guides
165-4130Criterion™ Dodeca™ Cell
170-4070 Criterion Blotter with Plate Electrodes
170-4071 Criterion Blotter with Wire Electrodes
170-4155Trans-Blot® Turbo™ Transfer Starter System
170-3940 Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell
164-5050 PowerPac™ Basic Power Supply
164-5052 PowerPac HC High Current Power Supply
170-8270
Gel Doc™ EZ Imaging System
170-8274Stain-Free™ Sample Tray
48
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Catalog # Description
Protein Standards
161-0363 Precision Plus Protein™ Unstained Standards (10–250 kD), 1 ml, 100 applications
161-0373 Precision Plus Protein All Blue Prestained Standards (10–250 kD), 500 μl,
50 applications
161-0374 Precision Plus Protein Dual Color Standards (10–250 kD), 500 μl, 50 applications
161-0375 Precision Plus Protein™ Kaleidoscope™ Standards (10–250 kD), 500 μl, 50 applications
161-0376 Precision Plus Protein™ WesternC™ Standards (10–250 kD), 250 μl, 50 applications
161-0377
Precision Plus Protein Dual Xtra Standards (2.5–250 kD), 500 μl, 50 applications
161-0385 Precision Plus Protein WesternC Pack (10–250 kD), 50 applications each of standard and StrepTactin-HRP
161-0317 SDS-PAGE Standards, broad range, 200 μl
Premixed Running Buffers
161-0732 10x Tris/Glycine/SDS, 1 L
161-0772 10x Tris/Glycine/SDS, 5 L
161-0734 10x Tris/Glycine, 1 L
161-0744 10x Tris/Tricine/SDS, 1 L
161-0788 XT MOPS Running Buffer, 20x, 500 ml
161-0789 XT MES Running Buffer, 20x, 500 ml
161-0790 XT Tricine Running Buffer, 20x, 500 ml
161-0793 XT MOPS Buffer Kit, includes 500 ml 20x XT MOPS running buffer, 10 ml 4x XT sample
buffer, 1 ml 20x XT reducing agent
161-0796 XT MES Buffer Kit, includes 500 ml 20x XT MOPS running buffer, 10 ml 4x XT sample
buffer, 1 ml 20 x XT reducing agent
161-0797 XT Tricine Buffer Kit, includes 500 ml 20x XT MOPS running buffer, 10 ml 4x XT sample
buffer, 1 ml 20x XT reducing agent
161-0761 10x IEF Anode Buffer, 250 ml
161-0762 10x IEF Cathode Buffer, 250 ml
161-0733
10x Tris/Boric Acid/EDTA, 1 L
161-0770 10x Tris/Boric Acid/EDTA, 5 L
161-0765 Zymogram Renaturation Buffer, 125 ml
161-0766 Zymogram Development Buffer, 125 ml
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
49
Criterion Precast Gels
Catalog # Description
Premixed Sample Buffers
161-0737 Laemmli Sample Buffer, 30 ml1
161-0738 Native Sample Buffer, 30 ml
161-0791 XT Sample Buffer, 4x, 10 ml
161-0792 XT Reducing Agent, 20x, 1 ml
161-0739 Tricine Sample Buffer, 30 ml
161-0763 IEF Sample Buffer, 30 ml
161-0764 Zymogram Sample Buffer, 30 ml
161-0767 Nucleic Acid Sample Buffer, 5x, 10 ml
161-0768 TBE-Urea Sample Buffer, 30 ml
Component Reagents
161-0719 Tris, 1 kg
161-0718
Glycine, 1 kg
161-0301
SDS, 100 g
161-0416 SDS Solution, 10% (w/v), 250 ml
166-2404
10% Tween 20, 5 ml
170-6404
Blotting-Grade Blocker, 300 g
161-0710 2-Mercaptoethanol, 25 ml
161-0611 Dithiothreitol (DTT), 5 g
161-0404 Bromophenol Blue, 10 g
Total Protein Gel and Blot Stains
161-0786 Bio-Safe™ Coomassie Stain, 1 L
161-0400 Coomassie Brilliant Blue R-250, 10 g
161-0436 Coomassie Blue R-250 Stain Solution, 1 L
161-0438 Coomassie Blue R-250 Destain Solution, 1 L
161-0443 Silver Stain Kit
161-0449 Silver Stain Plus™ Kit
170-6527 Colloidal Gold Total Protein Stain, 500 ml
161-0440 Zinc Stain and Destain Kit
170-3127 SYPRO Ruby Protein Blot Stain, 200 ml
161-0491 Flamingo™ Fluorescent Gel Stain (10x), 100 ml
161-0496 Oriole™ Fluorescent Protein Gel Stain (1x), 1 L
1
50
May require addition of 2-mercaptoethanol or DTT
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Catalog # Description
Immunoblot Detection Reagents
161-0385
Precision Plus Protein™ WesternC™ Pack
170-5070 Immun-Star™ WesternC™ Chemiluminescent Kit, 100 ml
170-6431 HRP Conjugate Substrate Kit, 4CN
170-6535 HRP Color Development Reagent, DAB, 5 g
170-8238 Amplified Opti-4CN™ Substrate Kit
170-8235 Opti-4CN Substrate Kit
170-6432 AP Conjugate Substrate Kit
170-5012 Immun-Star™ Substrate Pack
Blotting Membranes
162-0232 0.2 μm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
162-0233 0.2 μm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
162-0234 0.45 μm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
162-0235 0.45 μm Nitrocellulose/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
162-0236 Sequi-Blot™ PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 20 pack
162-0237 Sequi-Blot PVDF/Filter Paper Sandwich, 8.5 x 13.5 cm, 50 pack
170-4157
Trans-Blot Turbo Midi PVDF Transfer Packs
170-4159
Trans-Blot Turbo Midi Nitrocellulose Transfer Packs
For additional product sizes, please refer to the Bio-Rad catalog or website.
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
51
Criterion Precast Gels
52
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
Instruction Manual and Application Guide
Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
53
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad .com USA 800 424 6723 Australi a 61 2 9914 2800 Austri a 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600
Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finlan d 09 804 22 00
France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungar y 36 1 459 6100 India 91 124 4029300
Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Malaysia 60 3 2117 5260 Mexico 52 555 488 7670
The Netherland s 0318 540666 New Zealan d 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapor e 65 6415 3170 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700
Switzerlan d 061 717 95 55 Taiwan 886 2 2578 7189 Thailan d 66 2 6518311 United Kingdom 020 8328 2000
Life Science
Group
4110001 Rev F
US/EG
1011
Sig 0211
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement