Ian M. Rosenberg

Protein Analysis and Purification

Benchtop Techniques

• Broschiertes Buch

Produktbeschreibung

Bergey’s Manual of Systematic Bacteriology, one of the most comprehensive and authoritative works in the field of prokaryotic systematics is undergoing an extensive revision that will ultimately culminate in a five volume Second Edition. Arrangement of the content of the Second Edition follows the now familiar and well regarded phylogeny of the 16S rRNA gene, yet retains much of the layout of the First Edition. Volume 1, encompassing the Archaea, Deeply Branching and Phototrophic Bacteria was published in 2001. We are pleased to announce that work on Volume 2, The Proteobacteria, has been completed. This culminates a four year effort by Bergey's Manual Trust and more than 150 internationally recognized authorities to provide a comprehensive view of the Proteobacteria, the largest prokaryotic phylum.

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How one goes about analyzing proteins is a constantly evolving field that is no longer solely the domain of the protein biochemist. Investi gators from diverse disciplines find themselves with the unanticipated task of identifying and analyzing a protein and studying its physical properties and biochemical interactions. In most cases, the ultimate goal remains understanding the role(s) that the target protein is playing in cellular physiology. It was my intention that this manual would make the initial steps in the discovery process less time consuming and less intimidating. This book is not meant to be read from cover to cover. The expanded Table of Contents and the index should help locate what you are seeking. My aim was to provide practically oriented information that will assist the experimentalist in benchtop problem solving. The appendices are filled with diverse information gleaned from catalogs, handbooks, and manuals that are presented in a distilled fashion designed tosave trips to the library and calls to technical service representatives. The user is encouraged to expand on the tables and charts to fit individual experimental situations. This second edition pays homage to the computer explosion and the various genome projects that have revolutionized how benchtop scientific research is performed. Bioinformatics and In silica science are here to stay. However, the second edition still includes recipes for preparing buffers and methods for lysing cells.

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Produktdetails

• Verlag: Birkhäuser / Birkhäuser Boston / Springer, Berlin
• Artikelnr. des Verlages: 10988541, 978-0-8176-4341-6
• 2nd ed.
• Seitenzahl: 520
• Erscheinungstermin: 29. September 2004
• Englisch
• Abmessung: 249mm x 186mm x 29mm
• Gewicht: 942g
• ISBN-13: 9780817643416
• ISBN-10: 0817643419
• Artikelnr.: 13128234

Autorenporträt

Ian M. Rosenberg, Massachusetts General Hospital, Boston, USA

Inhaltsangabe

1 An Overview of This Manual.- Kits, Cores, and Computers.- How to Use This Book.- Basic Laboratory Equipment.- Laboratory Automation.- Beyond Protein Analysis and Purification.- 2 Protein Structure.- A. The Amino Acids.- B. The Four Levels of Protein Structure.- C. Chemical Characteristics of Proteins.- 3 Tracking the Target Protein.- A. Labeling Cells and Proteins.- B. Lysis-Preparation of the Cell Free Extract.- C. Principles of Immunoprecipitation.- D. Additional Methods to Identify Associated Proteins.- 4 Electrophoretic Techniques.- to Polyacrylamide Gel Electrophoresis (PAGE).- A. Preparation of SDS-Polyacrylamide Gels.- B. 2-Dimensional (2-D) Gel Systems.- D. Recovery of Proteins from the Gel.- E. Identification of Enzyme Activity in Polyacrylamide Gels.- 5 Getting Started with Protein Purification.- A. Making a Cell Free Extract.- B. Protein Quantitation.- C. Manipulating Proteins in Solution.- D. Precipitation Techniques.- 6 Membrane Proteins.- A. Peripheral Membrane Proteins.- B. Integral Membrane Proteins.- C. Detergents.- 7 Transfer and Detection of Proteins on.- Membrane Supports.- A. Transfer of Proteins to Membrane Supports.- B. Staining the Blot.- C. Recovery of Proteins from the Blot.- D. Detection of the Target Protein.- 8 Identification of the Target Protein.- A. Peptide Mapping.- B. Enzymatic Cleavage of Proteins.- C. Chemical Cleavage.- D. Microsequencing from PVDF Membranes.- MS Basics.- Protein Identification by MS.- Protein Database Searches.- The Rise of Biological Databases.- 9 Identifying and Analyzing Posttranslational.- Modifications.- A. Glycosylation.- B. Phosphorylation.- C. Lipid Modification of Proteins.- D. Selected Modifications.- 10 Chromatography.- A. Important Terminology Used in Chromatography.- B. Gel FiltrationChromatography.- C. Introduction to HPLC.- D. Ion Exchange Chromatography: Separation on the Basis of Charge.- E. Hydrophobic Interaction Chromatography (HIC).- Flow Rate.- 11 Recombinant Protein Techniques.- Recombinant Protein for Antibody Production.- Protein for Biochemical or Cell Biological Studies.- A. In vitro Transcription and Translation.- B. Recombinant Gene Products in E. coli: Expression, Identification and Characterization.- D. Expression of Foreign Proteins in Eukaryotic Cells.- Appendices.- A. Safety Considerations.- First Aid: Emergency Procedures.- B. Antibody Preparation.- Production of Polyclonal Antisera in Rabbits.- Purification of Antibody Using Protein A Affinity Columns.- Numbering Mice.- Molarity.- Choosing and Preparing Buffers.- Common Laboratory Solutions.- Extinction Coefficients.- D. Nucleic Acids.- Spectrophotometric Conversions.- DNA/Protein Conversions.- Oligonucleotide Concentrations.- RNA Precipitation.- E. Modifications and Motifs.- Nomenclature.- Protein Modification Sequences.- Protein Kinase Recognition Sequence Motifs.- Subcellular Localization Motifs.- Protein Databases.- F. Centrifugation.- Nomogram.- General Purpose Centrifuge Rotors.- Ultracentrifuge Rotors.- G. Proteases and Proteolytic Enzyme Inhibitors.- Commonly Used Proteases.- Protease Inhibitors.- H. Radioactivity.- Manual and Machine Film Processing.- L Tissue Culture.- Transwell Permeable Supports.- J. Miscellaneous.- Unit Prefixes.- The Greek Alphabet.- Abbreviations.- HPLC Pump Pressure Conversion.- Dipeptide Masses.- Mass Differences Considered in Molecular Weight Analysis of Proteins.- K. List of Suppliers, Vendors, Manufacturers.

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An Overview of This Manual.- Protein Structure.- Tracking the Target Protein.- Electrophoretic Techniques.- Getting Started with Protein Purification.- Membrane Proteins.- Transfer and Detection of Proteins on Membrane Supports.- Identification of the Target Protein.- Identifying and Analyzing Posttranslational Modifications.- Chromatography.- Recombinant Protein Techniques.