Elsevier S & T

Handbook of Isoelectric Focusing and Proteomics

Garfin, David; Ahuja, Satinder

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Table of contents
  • CONTENTSv
  • PREFACExi
  • CONTRIBUTORSxv
  • INCIDENTS OF TRAVEL IN IEF AND IPGSxvii
  • Chapter 1. Overview1
  • I. Separations by IEF2
  • II. Evolution and Development of IEF4
  • III. Theory and Simulation of IEF4
  • IV. Generation of pH Gradients5
  • V. Slab Gel IEF5
  • VI. Two-dimensional Gel Electrophoresis (2-DE)6
  • VII. Practices and Pitfalls of Sample Preparation6
  • VIII. Protein Detection and Imaging7
  • IX. Capillary IEF7
  • X. Preparative IEF8
  • XI. IEF and Proteomics9
  • XII. Chromatofocusing10
  • XIII. Alternate Electrofocusing Methods11
  • XIV. Summary12
  • References12
  • Chapter 2. Evolution and Development of Isoelectric Focusing13
  • I. Introduction13
  • II. The Rise of Electrophoresis13
  • III. Kolins Isoelectric SpectraŽ„The Artificial pH Gradient18
  • IV. Svenssons IEF„Vesterbergs Synthesis21
  • V. Progress in Preparative and Analytical IEF26
  • VI. The Immobilized pH Gradients28
  • VII. Two-dimensional Gel Electrophoresis and Blotting of Proteins30
  • VIII. Capillary IEF31
  • IX. Special Features in the Practice and Theory of IEF31
  • X. Reviews on IEF32
  • XI. Concluding Remarks33
  • References34
  • Chapter 3. Theory and Simulation of Isoelectric Focusing41
  • I. Principles of Isoelectric Focusing41
  • II. Numerical Simulation of IEF51
  • III. Illustrative Simulations of IEF57
  • IV. Summary66
  • References67
  • Chapter 4. Generation of pH Gradients69
  • I. Introduction69
  • II. pH Gradients in the Early History of IEF70
  • III. The Development of Carrier Ampholytes71
  • IV. Practical Aspects of Carrier Ampholyte-generated pH Gradients75
  • V. Limitations of the Carrier Ampholyte Method78
  • VI. Early Alternative IEF Modes Not Requiring Carrier Ampholytes79
  • VII. Immobilized pH Gradients81
  • VIII. Use of Immobilized Buffers in Preparative IEF84
  • IX. Practical Aspects of Immobilized pH Gradients85
  • References87
  • Chapter 5. Slab Gel IEF93
  • I. Introduction93
  • II. Equipment95
  • III. The Gel Matrix97
  • IV. Polyacrylamide Gels98
  • V. Agarose Gels108
  • VI. Dextran Gels110
  • VII. Experimental Protocols: Polyacrylamide Slab Gel IEF111
  • References120
  • Chapter 6. Two-Dimensional GeI EIectrophoresis123
  • I. Introduction123
  • II. Equilibration of First Dimension IEF Gels124
  • III. SDS–Page128
  • IV. Protein Detection133
  • V. Gel Reproducibility137
  • VI. Practical Applications138
  • VII. Advantages and Limitations of 2-DE140
  • VIII. Summary140
  • References140
  • Chapter 7. Some Practices and Pitfalls of Sample Preparation for lsoelectric Focusing in Proteomics147
  • I. Introduction147
  • II. Reduction and Alkylation150
  • III. Beta Elimination of Cysteine152
  • IV. Carbamylation155
  • V. Stable Isotope Labeling-based Quantitation157
  • VI. Sample Homogenization and Nucleic Acid Removal157
  • VII. Membrane Proteins160
  • References162
  • Chapter 8. Protein Detection and Imaging in IEF Gels165
  • I. Introduction165
  • II. Organic Dye Staining166
  • III. Silver Staining167
  • IV. Reverse Staining169
  • V. Fluorescence Staining169
  • VI. Label-less Detection172
  • VII. Post-translational Modification Detection172
  • VIII. Acquiring Images from Stained Gels173
  • IX. Conclusion176
  • Acknowledgements176
  • References176
  • Chapter 9. Capillary lsoelectric Focusing181
  • I. Introduction181
  • II. Sample Preparation183
  • III. Ampholyte Selection and Sample Introduction185
  • IV. Focusing186
  • V. Mobilization Techniques187
  • VI. Capillary Selection193
  • VII. Minimizing Protein Precipitation195
  • VIII. Internal Standards for cIEF195
  • IX. Imaging cIEF196
  • X. cIEF-Mass Spectrometry197
  • XI. cIEF In Microchannels199
  • XII. Applications of cIEF200
  • References205
  • Chapter 10. Free-Flow lsoelectric Focusing211
  • I. Introduction211
  • II. Principle of FFE212
  • III. Instrumentation220
  • IV. Applications231
  • V. Summary239
  • References239
  • Chapter 11. lsoelectric Focusing and Proteomics247
  • I. Introduction247
  • II. The Proteomics Workflow249
  • III. IEF for Prefractionation252
  • IV. IEF in Two-dimensional Electrophoresis254
  • V. Conclusions259
  • Glossary259
  • Acknowledgments259
  • References260
  • Chapter 12. Chromatofocusing265
  • I. Introduction265
  • II. Conventional Chromatofocusing (Internal pH Gradient Generation)267
  • III. Gradient Chromatofocusing (External pH Gradient Generation)277
  • IV. Performance Characteristics283
  • V. Applications288
  • References290
  • Chapter 13. Alternative Electrofocusing Methods297
  • I. Introduction297
  • II. Theory301
  • III. Results311
  • IV. Discussion315
  • V. Conclusion317
  • Acknowledgments317
  • References318
  • Index321
Book details
  • Vendor Elsevier S & T
  • SKU 9780120887521
  • ISBN-13 9780080533629
  • Author Garfin, David; Ahuja, Satinder
  • Category Science
  • Subject Analytic

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Isoelectric focusing (IEF) is a high-resolution, stand-alone technique that can be used as an analytical method or tool for protein purification. The only current book on the market, the Handbook of Isoelectric Focusing and Proteomics is the ideal 'one-stop' source for germane information in this discipline. This highly practical book also contains chapters on alternative methods that may pave the way in the search for efficient techniques for fractionating and purifying proteins. Complete with the history of IEF focusing to authors' insights and practical tips, this book is a must for anyone working in proteomics.

* Is the only current book available on the subject
* Includes author insights and practical tips
* Is an ideal single source for students and researchers working in proteomics