2D PAGE: Sample Preparation and Fractionation

Focusing on two-dimensional electrophoresis (2-DE), this book sets forth the principles and recent developments of sample preparation and fractionation tools in expression proteomics. 2-DE, with its unique capacity to resolve thousands of proteins in a single run, remains a fundamental research tool for nearly all protein-related scientific projects. Step-by-step instructions alongside detailed illustrations enable biochemists, molecular and cellular biologists, and protein scientists to quickly learn how to perform the methods. Tricks of the trade and notes on troubleshooting and avoiding pitfalls help ensure successful results every time. The methods covered in this book are not limited to 2-DE; they can also be applied to a broad range of protein separation techniques.

Contains step-by-step instructions that ensure successful results Provides tricks of the trade and notes on troubleshooting and avoiding known pitfalls

Klappentext

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 preparation 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.

Invaluable for students of Biochemistry, Biomedicine, Biology and Genomics and essential for the practicing scientists, *2D PAGE: Sample Preparation and Fractionation, Volumes 1 & 2 *delve into the study of proteins with comprehensive and in depth methods and protocols to provide a greater understanding of this intricate and complicated field.

Inhalt
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).- ICPLIsotope-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.