Handbook of Isoelectric Focusing and

The emergence of proteomics as a mature discipline has brought renewed interest in isoelectric focusing (IEF), as it is a rapid and reliable technique that provides high resolution for protein fractionation. The Handbook of Isoelectric Focusing and Proteomics is intended to serve as a single source for all relevant information on IEF.

PREFACE

xi

CONTRIBUTORS

xv

INCIDENTS OF TRAVEL IN IEF AND IPGS

xvii

PIER GIORGIO RIGHETTI

1. Overview

DAVID E. GARFIN AND SATINDER AHUJA

I. Separations by IEF

2

(2)

II. Evolution and Development of IEF

4

(1)

III. Theory and Simulation of IEF

4

(1)

IV. Generation of pH Gradients

5

(1)

V. Slab Gel IEF

5

(1)

VI. Two-dimensional Gel Electrophoresis (2-DE)

6

(1)

VII. Practices and Pitfalls of Sample Preparation

6

(1)

VIII. Protein Detection and Imaging

7

(1)

IX. Capillary IEF

7

(1)

X. Preparative IEF

8

(1)

XI. IEF and Proteomics

9

(1)

XII. Chromatofocusing

10

(1)

XIII. Alternate Electrofocusing Methods

11

(1)

XIV. Summary

12

(1)

References

12

(1)

2. Evolution and Development of Isoelectric Focusing

ÁKOS VÉGVÁRI AND FERENC KILÁR

I. Introduction

13

(1)

II. The Rise of Electrophoresis

13

(5)

III. Kolin's "Isoelectric Spectra"-The Artificial pH Gradient

18

(3)

IV. Svensson's IEF-Vesterberg's Synthesis

21

(5)

V. Progress in Preparative and Analytical IEF

26

(2)

VI. The Immobilized pH Gradients

28

(2)

VII. Two-dimensional Gel Electrophoresis and Blotting of Proteins

30

(1)

VIII. Capillary IEF

31

(1)

IX. Special Features in the Practice and Theory of IEF

31

(1)

X. Reviews on IEF

32

(1)

XI. Concluding Remarks

33

(1)

References

34

(7)

3. Theory and Simulation of Isoelectric Focusing

T.L. SOUNART, PA. SAFIER, AND J.C. BAYGENTS

I. Principles of Isoelectric Focusing

41

(10)

II. Numerical Simulation of IEF

51

(6)

III. Illustrative Simulations of IEF

57

(9)

IV. Summary

66

(1)

References

67

(2)

4. Generation of pH Gradients

TOM BERKELMAN

I. Introduction

69

(1)

II. pH Gradients in the Early History of IEF

70

(1)

III. The Development of Carrier Ampholytes

71

(4)

IV. Practical Aspects of Carrier Ampholyte-generated pH Gradients

75

(3)

V. Limitations of the Carrier Ampholyte Method

78

(1)

VI. Early Alternative IEF Modes Not Requiring Carrier Ampholytes

79

(2)

VII. Immobilized pH Gradients

81

(3)

VIII. Use of Immobilized Buffers in Preparative IEF

84

(1)

IX. Practical Aspects of Immobilized pH Gradients

85

(2)

References

87

(6)

5. Slab Gel IEF

REINER WESTERMEIER

I. Introduction

93

(2)

II. Equipment

95

(2)

III. The Gel Matrix

97

(1)

IV. Polyacrylamide Gels

98

(10)

V. Agarose Gels

108

(2)

VI. Dextran Gels

110

(1)

VII. Experimental Protocols: Polyacrylamide Slab Gel IEF

111

(9)

References

120

(3)

6. Two-Dimensional Gel Electrophoresis

MARK P. MOLLOY AND MICHAEL T. McDOWELL

I. Introduction

123

(1)

II. Equilibration of First Dimension IEF Gels

124

(4)

III. SDS-Page

128

(5)

IV. Protein Detection

133

(4)

V. Gel Reproducibility

137

(1)

VI. Practical Applications

138

(2)

VII. Advantages and Limitations of 2-DE

140

(1)

VIII. Summary

140

(1)

References

140

(7)

7. Some Practices and Pitfalls of Sample Preparation for Isoelectric Focusing in Proteomics

BEN HERBERT

I. Introduction

147

(3)

II. Reduction and Alkylation

150

(2)

III. Beta Elimination of Cysteine

152

(3)

IV. Carbamylation

155

(2)

V. Stable Isotope Labeling-based Quantitation

157

(1)

VI. Sample Homogenization and Nucleic Acid Removal

157

(3)

VII. Membrane Proteins

160

(2)

References

162

(3)

8. Protein Detection and Imaging in IEF Gels

WAYNE F. PATTON

I. Introduction

165

(1)

II. Organic Dye Staining

166

(1)

III. Silver Staining

167

(2)

IV. Reverse Staining

169

(1)

V. Fluorescence Staining

169

(3)

VI. Label-less Detection

172

(1)

VII. Post-translational Modification Detection

172

(1)

VIII. Acquiring Images from Stained Gels

173

(3)

IX. Conclusion

176

(1)

Acknowledgements

176

(1)

References

176

(5)

9. Capillary Isoelectric Focusing

TIM WEHR

I. Introduction

181

(2)

II. Sample Preparation

183

(2)

III. Ampholyte Selection and Sample Introduction

185

(1)

IV. Focusing

186

(1)

V. Mobilization Techniques

187

(6)

VI. Capillary Selection

193

(2)

VII. Minimizing Protein Precipitation

195

(1)

VIII. Internal Standards for cIEF

195

(1)

IX. Imaging cIEF

196

(1)

X. cIEF-Mass Spectrometry

197

(2)

XI. cIEF In Microchannels

199

(1)

XII. Applications of cIEF

200

(5)

References

205

(6)

10. Free-Flow Isoelectric Focusing

PETER J.A. WEBER, GERHARD WEBER, CHRISTOPH ECKERSKORN, ULRICH SCHNEIDER, AND ANTON POSCH

I. Introduction

211

(1)

II. Principle of FFE

212

(8)

III. Instrumentation

220

(11)

IV. Applications

231

(8)

V. Summary

239

(1)

References

239

(8)

11. Isoelectric Focusing and Proteomics

MELANIE Y. WHITE AND STUART J. CORDWELL

I. Introduction

247

(2)

II. The Proteomics Workflow

249

(3)

III. IEF for Prefractionation

252

(2)

IV. IEF in Two-dimensional Electrophoresis

254

(5)

V. Conclusions

259

(1)

Glossary

259

(1)

Acknowledgments

259

(1)

References

260

(5)

12. Chromatofocusing

DAVID ANDERSON

I. Introduction

265

(2)

II. Conventional Chromatofocusing (Internal pH Gradient Generation)

267

(10)

III. Gradient Chromatofocusing (External pH Gradient Generation)

277

(6)

IV. Performance Characteristics

283

(5)

V. Applications

288

(2)

References

290

(7)

13. Alternative Electrofocusing Methods

CORNELIUS F. IVORY

I. Introduction

297

(4)

II. Theory

301

(10)

III. Results

311

(4)

IV. Discussion

315

(2)

V. Conclusion

317

(1)

Acknowledgments

317

(1)

References

318

(3)

Index

321

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Handbook of Isoelectric Focusing and Proteomics

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