Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods

1 Introduction: The Virtue of Proteomics.- 2 Solubilization of Proteins in 2D Electrophoresis.- 1 Introduction.- 2 Rationale of Solubilization-Breaking Molecular Interactions.- 3 Initial Solubilization.- 4 Solubility During IEF.- 5 Conclusions: Current Limits and How to Push Them.- References.- 3 Two-Dimensional Electrophoresis with Carrier Ampholytes.- 1 Introduction.- 2 Sample Preparation.- 3 First Dimension. Standard Isoelectric Focusing.- 4 Second Dimension. Standard Slab Gel Electrophoresis.- 5 Modification of the Standard Two-Dimensional Gel Method.- 6 Visualization of Separated Proteins: Silver Staining.- References.- Appendix: Problems and Troubleshooting.- 4 Two-Dimensional Electrophoresis with Immobilized pH Gradients.- 1 Introduction.- 2 Sample Preparation.- 3 First Dimension: IEF with IPGs.- 4 Equilibration of IPG Strips.- 5 Second Dimension: SDS-PAGE.- References.- Appendix A: General Troubleshooting.- Appendix B: Troubleshooting for IPG and Horizontal PAGE.- 5 Detection of Proteins on Two-Dimensional Electrophoresis Gels.- 1 Introduction.- 2 Detection by Organic Dyes.- 3 Detection by Differential Precipitation of Salts.- 4 Detection by Metal Ion Reduction (Silver Staining).- 5 Detection by Fluorescence.- 6 Detection of Radioactive Isotopes.- 7 Conclusions and Future Prospects.- References.- 6 Blotting and Immunoaffinity Identification of Two-Dimensional Electrophoresis-Separated Proteins.- 1 Introduction.- 2 Protein Transfer onto Membranes.- 3 Membrane Staining.- 4 Immunodetection.- 5 Assignment of Two-Dimensional Immunoreactive Spots by Matching.- References.- 7 Identification of Proteins by Amino Acid Composition After Acid Hydrolysis.- 1 Introduction.- 2 Blotting of Proteins from Gels to PVDF Membranes.- 3 Hydrolysis of PVDF-bound Proteins.- 4 Extraction of Amino Acids from PVDF Membranes.- 5 Derivatization and Chromatography.- 6 Amino Acid Analysis Troubleshooting Guide.- 7 Protein Identification by Database Matching.- 8 Identification by N-Terminal Sequence Tags and Amino Acid Composition.- 9 Conclusions.- References.- 8 Identification by Amino Acid Composition Obtained from Labeling.- 1 Introduction.- 2 Choice and Labeling Amino Acids.- 3 Single Labeling Method.- 4 Double Labeling Method Based on 35S Decay.- 5 Double Labeling Method using Scintillation Counting.- 6 Determination of pI and Mr.- 7 Construction of the Database.- 8 Search in a Protein Database.- 9 Result Analysis.- 10 Conclusion.- References.- 9 Identification of Proteins by Amino Acid Sequencing.- 1 Purpose of Protein Sequencing.- 2 N-Terminal Sequence Analysis by Edman Chemistry.- 3 Other Possibilities for Generation of Amino Acid Sequences.- 4 The Interface from Two-Dimensional Gel.- 5 Conclusion.- 6 Perspectives.- References.- 10 Identification of Proteins by Mass Spectrometry.- 1 Introduction.- 2 Protein Preparation Methods Compatible with MS.- 3 Enzymatic Digestion of Proteins.- 4 Sample Clean-up for ESI-MS and On-line Liquid Chromatography.- 5 Sample Clean-up for MALDI-MS.- 6 Mass Spectrometers.- 7 Protein Identification by Correlating MS DATA with Sequence Databases.- References.- 11 Mass Spectrometry of Intact Proteins from Two-Dimensional PAGE.- 1 Introduction.- 2 Elution of Proteins from Gels for Mass Spectrometry.- 3 Mass Spectrometry of Proteins Electroblotted onto Polymer Membranes.- 4 UV-MALDI Mass Spectrometry of Proteins Directly from Polyacrylamide Gels.- 5 Conclusion.- References.