Two-Dimensional Electrophoresis Protocols

by Sheehan, David; Tyther, Raymond

• ISBN13: 9781588299376
• ISBN10: 1588299376
• Format: Hardcover
• Copyright: 2009-06-30
• Publisher: Humana Pr Inc

Summary

Two-dimensional electrophoresis (2-DE) remains one of the most popular techniques for proteomic comparisons. Revolutionized by the development of immobilized pH gradient strips, this vital process has benefited greatly from recent developments in staining, mass spectrometry, and bioinformatics. In Two-Dimensional Electrophoresis Protocols, expert researchers examine these cutting edge proteomics methodologies, explore their application to a wide range of biological materials, and look at how these developments have helped standardize the design, execution, and analysis of proteomics experiments. Chapters reflect key steps in 2-DE experiments, including sample preparation, staining, post-translational modification, spot identification, and bioinformatics. Composed in the highly successful Methods in Molecular Biology series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls.Innovative and reader-friendly, Two-Dimensional Electrophoresis Protocols encourages newcomers to apply powerful 2-DE techniques to their own research, while also providing current and essential information for seasoned scientists.

Table of Contents

Preface	p. v
Contributors	p. xi
Overview Chapters
Two-Dimensional Electrophoresis: An Overview	p. 3
Solubilization of Proteins in 2DE: An Outline	p. 19
Selection of pH Ranges in 2DE	p. 31
Difficult Proteins	p. 47
Organelle Proteomics	p. 65
Applications of Chemical Tagging Approaches in Combination with 2DE and Mass Spectrometry	p. 83
Immunoblotting 2DE Membranes	p. 103
Protocols Chapters
Troubleshooting Image Analysis in 2DE	p. 113
Analysis of Bacterial Proteins by 2DE	p. 131
Proteomic Analysis of Caenorhabditis elegans	p. 145
Protein Extraction for 2DE	p. 171
Analysis of Proteins from Marine Molluscs	p. 197
Preparation and Analysis of Plastid Proteomes by 2DE	p. 205
High-Resolution 2DE	p. 221
Blue Native-Gel Electrophoresis Proteomics	p. 241
2DE for Proteome Analysis of Human Metaphase Chromosomes	p. 259
Microsomal Proteomics	p. 273
Prefractionation Using Microscale Solution IEF	p. 291
Diagonal Electrophoresis for Detection of Protein Disulphide Bridges	p. 305
High-Resolution Large-Gel 2DE	p. 311
Silver Staining of Proteins in 2DE Gels	p. 339
Detection of 4-Hydroxy-2-Nonenal- and 3-Nitrotyrosine-Modified Proteins Using a Proteomics Approach	p. 351
Proteomic Detection of Oxidized and Reduced Thiol Proteins in Cultured Cells	p. 363
Detection of Ubiquitination in 2DE	p. 377
Phosphoproteome Analysis by In-Gel Isoelectric Focusing and Tandem Mass Spectrometry	p. 383
Detection of Protein Glutathionylation	p. 397
Activity-Based Protein Profiling of Protein Tyrosine Phosphatases	p. 417
Active Protease Mapping in 2DE Gels	p. 431
Two-Dimensional Difference Gel Electrophoresis	p. 439
Protein Expression Profiling	p. 455
C-Terminal Sequence Analysis of 2DE-Separated Proteins	p. 469
Shotgun Protein Analysis by Liquid Chromatography-Tandem Mass Spectrometry	p. 483
De Novo Sequence Analysis of N-Terminal Sulfonated Peptides After in-Gel Guanidination	p. 495
Tryptic Digestion of In-Gel Proteins for Mass Spectrometry Analysis	p. 507
Database Interrogation Algorithms for Identification of Proteins in Proteomic Separations	p. 515
Creating 2DE Databases for the World Wide Web	p. 533
Index	p. 541
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