Handbook of Isoelectric Focusing and Proteomics (eBook)

Front Cover 1
Handbook of Isoelectric Focusing and Proteomics 4
Copyright Page 5
CONTENTS 6
PREFACE 12
CONTRIBUTORS 16
INCIDENTS OF TRAVEL IN IEF AND IPGS 18
Chapter 1. Overview 24
I. Separations by IEF 25
II. Evolution and Development of IEF 27
III. Theory and Simulation of IEF 27
IV. Generation of pH Gradients 28
V. Slab Gel IEF 28
VI. Two-dimensional Gel Electrophoresis (2-DE) 29
VII. Practices and Pitfalls of Sample Preparation 29
VIII. Protein Detection and Imaging 30
IX. Capillary IEF 30
X. Preparative IEF 31
XI. IEF and Proteomics 32
XII. Chromatofocusing 33
XIII. Alternate Electrofocusing Methods 34
XIV. Summary 35
References 35
Chapter 2. Evolution and Development of Isoelectric Focusing 36
I. Introduction 36
II. The Rise of Electrophoresis 36
III. Kolin’s “Isoelectric Spectra”—The Artificial pH Gradient 41
IV. Svensson’s IEF—Vesterberg‘s Synthesis 44
V. Progress in Preparative and Analytical IEF 49
VI. The Immobilized pH Gradients 51
VII. Two-dimensional Gel Electrophoresis and Blotting of Proteins 53
VIII. Capillary IEF 54
IX. Special Features in the Practice and Theory of IEF 54
X. Reviews on IEF 55
XI. Concluding Remarks 56
References 57
Chapter 3. Theory and Simulation of Isoelectric Focusing 64
I. Principles of Isoelectric Focusing 64
II. Numerical Simulation of IEF 74
III. Illustrative Simulations of IEF 80
IV. Summary 89
References 90
Chapter 4. Generation of pH Gradients 92
I. Introduction 92
II. pH Gradients in the Early History of IEF 93
III. The Development of Carrier Ampholytes 94
IV. Practical Aspects of Carrier Ampholyte-generated pH Gradients 98
V. Limitations of the Carrier Ampholyte Method 101
VI. Early Alternative IEF Modes Not Requiring Carrier Ampholytes 102
VII. Immobilized pH Gradients 104
VIII. Use of Immobilized Buffers in Preparative IEF 107
IX. Practical Aspects of Immobilized pH Gradients 108
References 110
Chapter 5. Slab Gel IEF 116
I. Introduction 116
II. Equipment 118
III. The Gel Matrix 120
IV. Polyacrylamide Gels 121
V. Agarose Gels 131
VI. Dextran Gels 133
VII. Experimental Protocols: Polyacrylamide Slab Gel IEF 134
References 143
Chapter 6. Two-Dimensional GeI EIectrophoresis 146
I. Introduction 146
II. Equilibration of First Dimension IEF Gels 147
III. SDS–Page 151
IV. Protein Detection 156
V. Gel Reproducibility 160
VI. Practical Applications 161
VII. Advantages and Limitations of 2-DE 163
VIII. Summary 163
References 163
Chapter 7. Some Practices and Pitfalls of Sample Preparation for lsoelectric Focusing in Proteomics 170
I. Introduction 170
II. Reduction and Alkylation 173
III. Beta Elimination of Cysteine 175
IV. Carbamylation 178
V. Stable Isotope Labeling-based Quantitation 180
VI. Sample Homogenization and Nucleic Acid Removal 180
VII. Membrane Proteins 183
References 185
Chapter 8. Protein Detection and Imaging in IEF Gels 188
I. Introduction 188
II. Organic Dye Staining 189
III. Silver Staining 190
IV. Reverse Staining 192
V. Fluorescence Staining 192
VI. Label-less Detection 195
VII. Post-translational Modification Detection 195
VIII. Acquiring Images from Stained Gels 196
IX. Conclusion 199
Acknowledgements 199
References 199
Chapter 9. Capillary lsoelectric Focusing 204
I. Introduction 204
II. Sample Preparation 206
III. Ampholyte Selection and Sample Introduction 208
IV. Focusing 209
V. Mobilization Techniques 210
VI. Capillary Selection 216
VII. Minimizing Protein Precipitation 218
VIII. Internal Standards for cIEF 218
IX. Imaging cIEF 219
X. cIEF-Mass Spectrometry 220
XI. cIEF In Microchannels 222
XII. Applications of cIEF 223
References 228
Chapter 10. Free-Flow lsoelectric Focusing 234
I. Introduction 234
II. Principle of FFE 235
III. Instrumentation 243
IV. Applications 254
V. Summary 262
References 262
Chapter 11. lsoelectric Focusing and Proteomics 270
I. Introduction 270
II. The Proteomics Workflow 272
III. IEF for Prefractionation 275
IV. IEF in Two-dimensional Electrophoresis 277
V. Conclusions 282
Glossary 282
Acknowledgments 282
References 283
Chapter 12. Chromatofocusing 288
I. Introduction 288
II. Conventional Chromatofocusing (Internal pH Gradient Generation) 290
III. Gradient Chromatofocusing (External pH Gradient Generation) 300
IV. Performance Characteristics 306
V. Applications 311
References 313
Chapter 13. Alternative Electrofocusing Methods 320
I. Introduction 320
II. Theory 324
III. Results 334
IV. Discussion 338
V. Conclusion 340
Acknowledgments 340
References 341
Index 344