2D Page

by Posch, Anton

• ISBN13: 9781588297228
• ISBN10: 1588297225
• Edition: 1st
• Format: Hardcover
• Copyright: 2008-04-30
• Publisher: Humana Pr Inc

Summary

The complex nature of proteins often requires a multitude of sample preparation options. In 2D PAGE: Sample Preparation and Fractionation, Volumes 1 & 2, Dr. Anton Posch presents a broad coverage of the principles and recent developments of sample prearation and fractionation tools in Expression Proteomics and specifically for two-dimensional electrophoresis (2-DE). Detailed laboratory methods describe the process of sample fractionation used to reduce the complexity of the protein sample and the methods of 2-DE analysis and a broad variety of gel-free mass spectrometry-based technologies to detect previously undetectable proteins. Vital information regarding protein isolation and preparation for reproducible proteome analysis previously overlooked by scientists has been clearly laid out in this pioneering two-volume set. Volume 1 focuses on the basics of sample preparation and protein labeling techniques with an emphasis on fractionation by chemical reagents, chromatography, and electrophoresis methods. The inclusion of meticulous enrichment strategies further aids the protein researcher.

Table of Contents

Preface	p. v
Contributors	p. xi
Sample Preparation Basics
Mechanical/Physical Methods of Cell Disruption and Tissue Homogenization	p. 3
Bacteria and Yeast Cell Disruption Using Lytic Enzymes	p. 23
Sample Solublization Buffers for Two-Dimensional Electrophoresis	p. 35
Quantitation of Protein in Samples Prepared for 2-D Electrophoresis	p. 43
Removal of Interfering Substances in Samples Prepared for Two-Dimensional (2-D) Electrophoresis	p. 51
Protein Concentration by Hydrophilic Interaction Chromatography Combined with Solid Phase Extraction	p. 63
Protein Labeling Techniques
Difference Gel Electrophoresis Based on Lys/Cys Tagging	p. 73
Isotope-Coded Two-Dimensional Maps: Tagging with Deuterated Acrylamide and 2-Vinylpyridine	p. 87
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)	p. 101
ICPL-Isotope-Coded Protein Label	p. 113
Radiolabeling for Two-Dimensional Gel Analysis	p. 125
Fractionation of Proteins by Chemical Reagents and Chromatography
Sequential Extraction of Proteins by Chemical Reagents	p. 139
Reducing Sample Complexity by RP-HPLC: Beyond the Tip of the Protein Expression Iceberg	p. 147
Enriching Basic and Acidic Rat Brain Proteins with Ion Exchange Mini Spin Columns Before Two-Dimensional Gel Electrophoresis	p. 157
Reducing the Complexity of the Escherichia coli Proteome by Chromatography on Reactive Dye Columns	p. 167
Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures	p. 187
Fractionation of Proteins by Immobilized Metal Affinity Chromatography	p. 205
Fractionation of Proteins by Heparin Chromatography	p. 213
Fractionation of Proteins by Electrophoresis Methods
Fractionation of Complex Protein Mixtures by Liquid-Phase Isoelectric Focusing	p. 225
Microscale Isoelectric Focusing in Solution	p. 241
Prefractionation, Enrichment, Desalting and Depleting of Low Volume and Low Abundance Proteins and Peptides Using the MF10	p. 257
Sample Prefractionation in Granulated Sephadex IEF Gels	p. 277
Free-Flow Electrophoresis System for Plasma Proteomic Applications	p. 287
Protein Fractionation by Preparative Electrophoresis	p. 301
Enrichment Strategies for Organelles, Multiprotein Complexes, and Specific Protein Classes
Isolation of Endocitic Organelles by Density Gradient Centrifugation	p. 317
Isolation of Highly Pure Rat Liver Mitochondria with the Aid of Zone-Electrophoresis in a Free Flow Device (ZE-FFE)	p. 333
Isolation of Proteins and Protein Complexes by Immunoprecipitation	p. 349
Isolation of Phosphoproteins	p. 365
Glycoprotein Enrichment Through Lectin Affinity Techniques	p. 373
Isolation of Bacterial Cell Membranes Proteins Using Carbonate Extraction	p. 397
Enrichment of Membrane Proteins by Partitioning in Detergent/Polymer Aqueous Two-Phase Systems	p. 403
The Isolation of Detergent-Resistant Lipid Rafts for Two-Dimensional Electrophoresis	p. 413
Isolation of Membrane Protein Complexes by Blue Native Electrophoresis	p. 423
Tissue Microdissection	p. 433
Index	p. 449
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