ELECTROPHORESIS Theory, Techniques

and Biochemical and Clinical Applications

SECOND EDITION

A N T H O N Y T. A N D R E W S

Food Research Institute, Reading

CLARENDON PRESS • OXFORD

CONTENTS

INTRODUCTION 1 1.1. Basic principles of electrophoresis 1

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE). HOMOGENEOUS GEL AND BUFFER SYSTEMS 5 2.1. The formation and structure of Polyacrylamide gel 5 2.2. Pore size effects 7 2.3. Some chemical properties of Polyacrylamide gel

constituents 8 2.4. Choosing a suitable gel and buffer system 9 2.5. Slabs or cylindrical gels? 12 2.6. The choice of apparatus and of setting up procedures 13 2.7. Typical gel formulations and pre-electrophoresis

treatments 19 2.8. Sample application and the electrophoresis 24 2.9. Gel staining methods 26 2.10. Destaining 43 2.11. Quantitative determination of separated components 44 2.12. Localization and quantitative measurement of

radioactively labelled components 51 2.13. Blotting techniques 59 2.14. Biochemical applications 74

POLYACRYLAMIDE GEL ELECTROPHORESIS USING MULTIPHASIC BUFFER SYSTEMS 79 3.1. The concentration and separation processes 80 3.2. The design of multiphasic buffer systems 81 3.3. Apparatus and setting-up procedures 83 3.4. Some typical gel and multiphasic buffer formulations 84 3.5. Sample application, electrophoresis, and analysis 86 3.6. Dissociating agents and disulphide bond cleaving

reagents 87 3.7. Omission of the stacking gel 88 3.8. Selective stacking and selective unstacking 89 3.9. Biochemical applications 90

CONTENTS

POLYACRYLAMIDE GEL ELECTROPHORESIS. MOLECULAR WEIGHT MEASUREMENT AND THE USE OF GEL CONCENTRATION GRADIENTS 93 4.1. The Ferguson plot 93 4.2. Alternative plots for estimation of molecular size 95 4.3. The use of the Ferguson plot in macromolecular

separations 98 4.4. Gels of graded porosity 100 4.5. The preparation and running of gradient gels 102 4.6. The estimation of molecular weights on gradient gels 108 4.7. Transverse gradient gel electrophoresis 110 4.8. Biochemical applications of PAGE in gels of differing

concentrations 113

POLYACRYLAMIDE GEL ELECTROPHORESIS IN THE PRESENCE OF DETERGENTS 117 5.1. The use of sodium dodecyl sulphate (SDS) for

molecular weight measurement 118 5.2. Theoretical background of molecular weight

measurement by SDS-PAGE 119 5.3. The standard method 123 5.4. Procedures using multiphasic buffers 124 5.5. Staining and destaining methods 126 5.6. The use of acrylamide concentration gradient gels 131 5.7. Separation of low-molecular-weight proteins 134 5.8. Peptide mapping of proteins by SDS-PAGE 136 5.9. The use of detergents other than SDS 138 5.10. Biochemical applications 144

ELECTROPHORESIS ON AGAROSE AND COMPOSITE POLYACRYLAMIDE-AGAROSE GELS. NUCLEIC ACID ANALYSIS 148 6.1. The preparation of agarose gels 149 6.2. The preparation of composite polyacrylamide-agarose

gels 152 6.3. Separation of nucleic acids 153 6.4. Measurement of sedimentation coefficients and

molecular weights 162 6.5. Restriction endonuclease maps 164 6.6. Sequencing of nucleic acids 166 6.7. Biochemical applications 173

CONTENTS xiii

7. PREPARATIVE GEL ELELECTROPHRESIS 178 7.1. Analysis of proteins without extraction from the

gel matrix 178 7.2. Small-scale preparations by extraction from

analytical-type gels 180 7.3. Apparatus for small-scale preparative experiments 196 7.4. Large-scale preparative methods with electrophoresis

over a constant path length 196 7.5. Apparatus for preparative electrophoresis over a

constant path length 199 7.6. Biochemical applications 201

8. IMMUNODIFFUSION AND IMMUNO­ELECTROPHORESIS 205 8.1. Immunodiffusion 205 8.2. Analysis of antigens by immunodiffusion techniques 207 8.3. Immuno-electrophoretic techniques 212 8.4. Enhancing the sensitivity of immunodiffusion and

immuno-electrophoresis experiments by staining and labelling techniques 226

8.5. The use of detergents 229 8.6. Immuno-electrofocusing 233 8.7. Affino-electrophoresis 234 8.8. Recovery of immunoprecipitates from gels 235 8.9. Biochemical applications 236

9. ISO-ELECTRIC FOCUSING 241 9.1. Iso-electric focusing: background and principles 241 9.2. Iso-electric focusing in density gradients 250 9.3. Iso-electric focusing in Polyacrylamide gels 258 9.4. Iso-electric focusing in agarose gels 282 9.5. Iso-electric focusing in beds of granulated gel 284 9.6. Biochemical applications 286

10. ISOTACHOPHORESIS (DISPLACEMENT ELECTROPHORESIS) 289 10.1. Isotachophoresis: principles and theory 289 10.2. Analytical isotachophoresis in capillary tubes 295 10.3. Other analytical and preparative isotachophoretic

methods 298 10.4. Biochemical applications 300

11.1. 11.2.

11.3. 11.4. 11.5.

11.6. 11.7. 11.8.

xiv CONTENTS

11. TWO-DIMENSIONAL ELECTROPHORESIS Two-stage separations Two-dimensional (2D) separations: General considerations and apparatus High resolution two-dimensional (2D)procedures "Low resolution" two-dimensional (2D) procedures High resolution non-denaturing or partially denaturing 2D procedures 2D separations of nucleic acids Staining, scanning, data handling etc of 2D gels Biochemical applications

12. MISCELLANEOUS ELECTROPHORESIS METHODS 12.1. Micro-gel electrophoresis methods 12.2. Gel electrophoresis in organic solvents 12.3. Starch gel electrophoresis 12.4. Paper electrophoresis 12.5. Thin-layer electrophoresis 12.6. Cellulose acetate 12.7. Zone electrophoresis in free solution: density

gradients and Sephadex columns 12.8. Affinity electrophoresis 12.9. Electrophoretic desorption in affinity chromatography

and in immuno-adsorption 12.10. Electrophoretic concentration

13. CLINICAL APPLICATIONS 13.1. Sample handling 13.2. Interpretation of the patterns of separated zones 13.3. Blood proteins 13.4. Other body fluids and tissues 13.5. Two-dimensional macromolecular maps

APPENDIX 1. METHODS FOR THE RADIOLABELLING OF PROTEINS AND NUCLEIC ACIDS

ALL Al.1.1. Al.1.2. Al.1.3. Al.1.4.

Proteins 125т a n d 13«!

Tritium (3H) I 4 C

32P, 35S, l4C, 3H, etc. by incubation methods

CONTENTS

A 1.2. Nucleic acids 404 Al.2.1. Incubation methods 404 Al.2.2. Chemical labelling with 125I 405 Al.2.3. Enzymic labelling with 32P 405

APPENDIX 2. ADDRESSES OF SOME SUPPLIERS OF ELECTROPHORESIS EQUIPMENT, CHEMICALS, AND RELATED REAGENTS 407

REFERENCES 409

GLOSSARY 438

INDEX 440