Protein Analysis and Purification

Benchtop Techniques

Häftad, Engelska, 2004

Av Ian M. Rosenberg, Ian M Rosenberg

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This comprehensive, practical, and user-friendly manual is designed to be a practical progression of experimental protocols that an inexperienced investigator may follow when embarking on a biochemical or biotechnology project. It will also appeal to experienced researches and graduate students as a benchtop handbook which, without sacrificing scientific concepts, maintains the spirit of experimentation.

Produktinformation

Utgivningsdatum2004-09-29
Mått178 x 254 x 29 mm
Vikt925 g
FormatHäftad
SpråkEngelska
Antal sidor520
Upplaga2
FörlagBirkhauser Boston Inc
ISBN9780817643416

Tillhör följande kategorier

Biovetenskap inom Naturvetenskap och teknik
Biokemi inom Naturvetenskap och teknik

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.