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This book, split into two volumes, presents a broad coverage of the principles and recent developments of sample preparation and fractionation tools in Expression Proteomics in general and for two-dimensional electrophoresis (2-DE) in particular. 2-DE, with its unique capacity to resolve thousands of proteins in a single run, is still a fundamental research tool for nearly all protein-related scientific projects. The methods described here in detail are not limited to 2-DE and can also be applied to other protein separation techniques. Because each biological sample is unique, a suited sample preparation strategy has to consider the type of sample as well as the type of biological question being addressed. The complex nature of proteins often requires a multitude of sample preparation options. In addition, sample preparation is not only a prerequisite for a successful and reproducible Proteomics exp- iment, but also the key factor to meaningful data evaluation. Interestingly, not much attention was paid to this area during Proteomics methodology dev- opment and therefore this book is intended to explain in depth how proteins from various sources can be properly isolated and prepared for reproducible Proteome analysis.
List of contents
Sample Preparation Basics.- Mechanical/Physical Methods of Cell Disruption and Tissue Homogenization.- Bacteria and Yeast Cell Disruption Using Lytic Enzymes.- Sample Solublization Buffers for Two-Dimensional Electrophoresis.- Quantitation of Protein in Samples Prepared for 2-D Electrophoresis.- Removal of Interfering Substances in Samples Prepared for Two-Dimensional (2-D) Electrophoresis.- Protein Concentration by Hydrophilic Interaction Chromatography Combined with Solid Phase Extraction.- Protein Labeling Techniques.- Difference Gel Electrophoresis Based on Lys/Cys Tagging.- Isotope-Coded Two-Dimensional Maps: Tagging with Deuterated Acrylamide and 2-Vinylpyridine.- Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).- ICPL-Isotope-Coded Protein Label.- Radiolabeling for Two-Dimensional Gel Analysis.- Fractionation of Proteins by Chemical Reagents and Chromatography.- Sequential Extraction of Proteins by Chemical Reagents.- Reducing Sample Complexity by RP-HPLC: Beyond the Tip of the Protein Expression Iceberg.- Enriching Basic and Acidic Rat Brain Proteins with Ion Exchange Mini Spin Columns Before Two-Dimensional Gel Electrophoresis.- Reducing the Complexity of the Escherichia coli Proteome by Chromatography on Reactive Dye Columns.- Reducing Sample Complexity in Proteomics by Chromatofocusing with Simple Buffer Mixtures.- Fractionation of Proteins by Immobilized Metal Affinity Chromatography.- Fractionation of Proteins by Heparin Chromatography.- Fractionation of Proteins by Electrophoresis Methods.- Fractionation of Complex Protein Mixtures by Liquid-Phase Isoelectric Focusing.- Microscale Isoelectric Focusing in Solution.- Prefractionation, Enrichment, Desalting and Depleting of Low Volume and Low Abundance Proteins and Peptides Using the MF10.-Sample Prefractionation in Granulated Sephadex IEF Gels.- Free-Flow Electrophoresis System for Plasma Proteomic Applications.- Protein Fractionation by Preparative Electrophoresis.- Enrichment Strategies for Organelles, Multiprotein Complexes, and Specific Protein Classes.- Isolation of Endocitic Organelles by Density Gradient Centrifugation.- Isolation of Highly Pure Rat Liver Mitochondria with the Aid of Zone-Electrophoresis in a Free Flow Device (ZE-FFE).- Isolation of Proteins and Protein Complexes by Immunoprecipitation.- Isolation of Phosphoproteins.- Glycoprotein Enrichment Through Lectin Affinity Techniques.- Isolation of Bacterial Cell Membranes Proteins Using Carbonate Extraction.- Enrichment of Membrane Proteins by Partitioning in Detergent/Polymer Aqueous Two-Phase Systems.- The Isolation of Detergent-Resistant Lipid Rafts for Two-Dimensional Electrophoresis.- Isolation of Membrane Protein Complexes by Blue Native Electrophoresis.- Tissue Microdissection.
Summary
This book, split into two volumes, presents a broad coverage of the principles and recent developments of sample preparation and fractionation tools in Expression Proteomics in general and for two-dimensional electrophoresis (2-DE) in particular. 2-DE, with its unique capacity to resolve thousands of proteins in a single run, is still a fundamental research tool for nearly all protein-related scientific projects. The methods described here in detail are not limited to 2-DE and can also be applied to other protein separation techniques. Because each biological sample is unique, a suited sample preparation strategy has to consider the type of sample as well as the type of biological question being addressed. The complex nature of proteins often requires a multitude of sample preparation options. In addition, sample preparation is not only a prerequisite for a successful and reproducible Proteomics exp- iment, but also the key factor to meaningful data evaluation. Interestingly, not much attention was paid to this area during Proteomics methodology dev- opment and therefore this book is intended to explain in depth how proteins from various sources can be properly isolated and prepared for reproducible Proteome analysis.
