Bulletin 2895

A Guide to Transfer and Detection
Third Edition

About This Manual
This reference manual provides information on the fundamentals of protein transfer and detection chemistries. It is also a guide to the methods, equipment, and reagents used in protein blotting experiments, and offers troubleshooting tips and technical advice. As a researcher using the blotting technique, you know that eachresearch objective, experimental approach, and protein sample can be different. Your equipment, sample, antibody, and detection chemistries all can impact your results. The goal of this manual is to provide you with a broad understanding of the variables you face when blotting, and how best to work with each of them to achieve the optimal results.

A History of Leadership and Quality
A pioneerin the design and manufacture of western blotting apparatus with 30 years of experience, Bio-Rad is considered the industry leader in providing high-quality, durable, and powerful blotting equipment. Bio-Rad offers superior products and expert technical service. Our goal is to support your research with the necessary tools and materials to optimize the analysis of complex protein samples.Meeting Your Blotting Needs
Turn to Bio-Rad for: • Gel-blotting equipment for an array of gel sizes • Microfiltration devices • Multiple-sample screening devices • Membranes for every binding requirement • Protein standards • Blotting buffers, reagents, and background removal kits • Colorimetric and chemiluminescent detection reagents • Total protein stains • Secondary antibodies and antibodyconjugates For detailed protocols on the use of any of the products mentioned in this guide, please refer to their instruction manuals, available in Adobe Acrobat (PDF) format at discover.bio-rad.com

Protein Blotting Guide

1

Table of Contents

Protein Blotting

Introduction to Protein Blotting

7

Power Conditions for Electrophoretic Transfers . . . . .21 Useful Equations . . . . . . . .. . . . . . . . . . . . . . . . . . .22 Joule Heating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Other Factors Affecting Transfer . . . . . . . . . . . . . . . .22 Relationship Between Power Settings and Transfer Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 High-Intensity Field Transfers Standard Field Transfers Selecting Power Supply Settings . . . . .. . . . . . . . . . .22 Transfers Under Constant Voltage Transfers Under Constant Current Transfers Under Constant Power

Chapter 1 Transfer
Transfer Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 Electrophoretic Transfer The Principle of Electrophoretic Transfer Types of Electrophoretic Transfer Microfiltration (Dot-Blotting) . . . . . . . . . . . . . . . . . . . .11Blotting Systems and Power Supplies . . . . . . . . . . . .12 Transfer Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . .12 Tank Blotting Apparatus Semi-Dry Blotting Apparatus Microfiltration Apparatus Power Supplies for Electrophoretic Transfers . . . . . . .15

General Guidelines for Transfer Buffers and Transfer Conditions . . . . . . . . . . . . . . . . . . . . . . .24

Chapter 3Performing the Transfer
Electrophoretic Transfer . . . . . . . . . . . . . . . . . . . . . . .25

Chapter 2 Membranes, Buffers, and Power Conditions
Membrane Selection . . . . . . . . . . . . . . . . . . . . . . . . . .17 Nitrocellulose and Supported Nitrocellulose . . . . . . . .17 Polyvinylidene Difluoride (PVDF) Membrane . . . . . . . .18

Performing a Tank Transfer . . . . . . . . . . . .. . . . . . . . .25 Preparing the Transfer Buffer, Gels, and Tank Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 Assembling the Gel and Membrane Sandwich . . . . . .26 Performing the Transfer . . . . . . . . . . . . . . . . . . . . . .27

Blotting Filter Paper . . . . . . . . . . . . . . . . . . . . . . . . . . .18

Performing a Semi-Dry Transfer . . . . . . . . . . ....