2D PAGE: Sample Preparation and Fractionation

Volume 1
2008
ISBN: 978-1-58829-722-8
Verlag: Humana Press

Buch, Englisch, Band 424, 459 Seiten, Format (B × H): 160 mm x 241 mm, Gewicht: 875 g

Reihe: Methods in Molecular Biology

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.

Zielgruppe

Professional/practitioner

Autoren/Hrsg.

Posch, Anton

Fachgebiete

• Naturwissenschaften Biowissenschaften Biochemie (nichtmedizinisch)
• Naturwissenschaften Chemie Organische Chemie Biochemie
• Naturwissenschaften Biowissenschaften Proteinforschung
• Medizin | Veterinärmedizin Medizin | Public Health | Pharmazie | Zahnmedizin Medizin, Gesundheitswesen Biomedizin, Medizinische Forschung, Klinische Studien
• Naturwissenschaften Biowissenschaften Biowissenschaften Genetik und Genomik (nichtmedizinisch)
• Naturwissenschaften Biowissenschaften Zellbiologie

Inhaltsverzeichnis

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.