Gel Electrophoresis of Proteins: A Practical Approach (Practical Approach Series)

373 Pages·1998·19.82 MB·English

by  B. D. Hames (Editor)

Gel Electrophoresis of Proteins
The Practical Approach Series
SERIES EDITOR
B. D. HAMES
School of Biochemistry and Molecular Biology
University of Leeds, Leeds LS2 9JT, UK
See also the Practical Approach web site at http://www.oup.co.uk/PAS
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Affinity Chromatography * Cell Separation
if Affinity Separations Cellular Calcium
Anaerobic Microbiology Cellular Interactions in
Development
Animal Cell Culture (2nd
edition) Cellular Neurobiology
Animal Virus Pathogenesis * Chromatin
Antibodies I and II Clinical Immunology
Antibody Engineering if Complement
Antisense Technology Crystallization of Nucleic
Acids and Proteins
if Applied Microbial Physiology
Cytokines (2nd edition)
Basic Cell Culture
The Cytoskeleton
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Pathology I and II
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DNA and Protein Sequence
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Biomechanics—Materials
DNA Cloning 1: Core
Biomechanics—Structures and Techniques (2nd edition)
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DNA Cloning 2: Expression
Biosensors Systems (2nd edition)
Carbohydrate Analysis (2nd DNA Cloning 3: Complex
edition) Genomes (2nd edition)
Cell-Cell Interactions DNA Cloning 4: Mammalian
The Cell Cycle Systems (2nd edition)
Cell Growth and Apoptosis Drosophila (2nd edition)
Electron Microscopy in Immunochemistry 2
Biology Immunocytochemistry
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Experimental Neuroanatomy
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Flow Cytometry (2nd edition) Medical Parasitology
Free Radicals Medical Virology
Gas Chromatography * MHC Volumes 1 and 2
Gel Electrophoresis of Nucleic if Molecular Genetic Analysis of
Acids (2nd edition) Populations (2nd edition)
Gel Electrophoresis of Molecular Genetics of Yeast
Proteins (3rd edition)
Molecular Imaging in
Gene Probes 1 and 2 Neuroscience
Gene Targeting Molecular Neurobiology
Gene Transcription Molecular Plant Pathology I
and II
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Molecular Virology
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HPLC of Macromolecules (2nd Neurochemistry (2nd edition)
edition) Neuronal Cell Lines
Human Cytogenetics I and II NMR of Biological
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Human Genetic Disease Non-isotopic Methods in
Analysis Molecular Biology
Immunochemistry 1 Nucleic Acid Hybridization
Oligonucleotides and Analogues Protein Function (2nd edition)
Oligonucleotide Synthesis Protein Phosphorylation
PCR1 Protein Purification
Applications
PCR2
Protein Purification Methods
Hybridization Protein Sequencing
Peptide Antigens Protein Structure (2nd edition)
Photosynthesis: Energy Protein Structure Prediction
Transduction Protein Targeting
Plant Cell Biology Proteolytic Enzymes
Plant Cell Culture (2nd edition) Pulsed Field Gel
Plant Molecular Biology Electrophoresis
Plasmids (2nd edition) RNA Processing I and II
Platelets * RNA-Protein Interactions
Postimplantation Mammalian * Signalling by Inositides
Embryos Subcellular Fractionation
Preparative Centrifugation Signal Transduction
Protein Blotting Transcription Factors
Protein Engineering Tumour Immunobiology
Gel Electrophoresis
of Proteins
A Practical Approach
THIRD EDITION
Edited by
B. D. HAMES
School of Biochemistry and Molecular Biology
University of Leeds, Leeds LS2 9JT, UK
Oxford New York Tokyo
OXFORD UNIVERSITY PRESS
1998
Oxford University Press, Great Clarendon Street, Oxford OX2 6DP
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A catalogue record for this book is available from the British Library
Library of Congress Cataloging in Publication Data
Gel electrophoresis of proteins: a practical approach/edited by
B.D. Homes.—3rd ed.
(Practical approach series; 197)
Includes bibliographical references and index.
1. Proteins—Analysis. 2. Gel electrophoresis. I. Homes, B.D.
II. Series.
QP551.G334 1998
572'.6—dc21 98-18884 CIP
ISBN 0 19 963641 9 (Hbk)
0 19 963640 0 (Pbk)
Typeset by Footnote Graphics, Warminster, Wilts
Printed in Great Britain by Information Press Ltd., Eynsham, Oxon
Preface
When the first edition of this book was published in 1981,1 had no idea that it
would lead to a major series of laboratory manuals, the Practical Approach
series, currently over 190 volumes in size and still growing. The popularity of
the series has led to second editions of many books. The second edition of Gel
Electrophoresls of Proteins: A Practical Approach was duly published in 1990.
Since that time, the field has continued to develop apace, so much so that some
current major techniques, such as capillary electrophoresis, were not even
mentioned in the second edition. Clearly, it is time for a third edition!
The goal in devising and editing the third edition of Gel Electrophoresis of
Proteins: A Practical Approach has been to reflect the laboratory usage of the
wide variety of methods that fall under this general subject heading. I make no
apology for revisiting the basic techniques of one-dimensional polyacrylamide
gel electrophoresis, including SDS-PAGE, since these methods have contin-
ued to be widely used on a regular basis in enormous numbers of laboratories
around the world, but even here there have been some very useful develop-
ments in technology. These are fully described in the first chapter which has
been written by new authors to bring fresh insights to the subject. Other tech-
niques have also undergone major developments in the intervening years, as
will be clear to readers of the chapters on isoelectric focusing, two-dimensional
gel electrophoresis, preparative gel electrophoresis, and polypeptide detection
systems. However, my aim was also to cover important emerging techniques.
Thus there are completely new chapters on capillary gel electrophoresis,
sequence analysis of gel-resolved proteins, fluorophore-labelled saccharide
electrophoresis, and analysis of protein:protein interactions by gel electro-
phoresis. In total, only four of the chapter subjects covered in the previous
edition are retained in the ten chapters of this new edition. Furthermore, eight
of the ten chapters in this book are written by new authors. Thus the third edi-
tion is in every sense a new text, and very definitely a major update in the field.
I am greatly indebted to the authors of each chapter who accepted with good
grace the very large number of editing changes I requested in that elusive quest
for perfection. Given that the authors are active researchers with intimate
knowledge of the methods (and pitfalls!) they describe, I am certain that the
book will help colleagues to apply the new methods successfully in their own
work. However, I have no illusions that this is the end of the story. The versatility
and power of gel electrophoretic techniques in biological research ensure that
still further developments and applications will continue to arise in future.
Leeds
January 1998 B. David Hames
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Contents
List of contributors xvii
Abbreviations xix
1. One-dimensional polyacrylamide gel
electrophoresis 1
Qinwei Shi and George Jackowski
1. Introduction 1
2. The polyacrylamide gel matrix 2
Chemical structure and mechanism of polymerization 2
Factors affecting polymerization 3
Resolving range of polyacrylamide gels 7
3. Gel apparatus 8
Electrophoresis apparatus 8
Power pack 10
Apparatus for gradient gels 10
Photopolymerization equipment 10
4. Choice of a gel system 11
Non-dissociating systems 11
Dissociating systems 11
Transverse gradient gels 13
5. SDS-polyacrylamide gel electrophoresis 13
Introduction 13
Reagent preparation 14
Gel preparation 16
Sample preparation 21
Sample loading and electrophoresis 27
Molecular mass estimation 29
Troubleshooting 33
6. Non-denaturing polyacrylamide gel electrophoresis 35
Introduction 35
Choice of buffers and polymerization catalyst 37
Preparation of non-denaturing slab gels 38
Sample preparation and electrophoresis of native proteins 39
7. Variations of standard polyacrylamide gel electrophoresis 40
Blue native polyacrylamide gel electrophoresis 40
Acid-urea polyacrylamide gel electrophoresis 42
Transverse gradient gel electrophoresis 42