Title:
Short protocols in protein science : a compendium of methods from current protocols in protein science
Publication Information:
Canada : John Wiley & Sons, 2003
ISBN:
9780471483380
Added Author:
Available:*
Library | Item Barcode | Call Number | Material Type | Item Category 1 | Status |
|---|---|---|---|---|---|
Searching... | 30000010063549 | QP551 S56 2003 | Open Access Book | Book | Searching... |
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Summary
Summary
Short Protocols in Protein Science provides condensed descriptions of more than 500 protocols compiled from Current Protocols in Protein Science. Drawing from both the original "core" manual as well as the quarterly update service, this compendium includes all step-by-step descriptions of the principal methods covered in Current Protocols in Protein Science .
Table of Contents
| Preface |
| Contributors |
| 1 Strategies of Protein Purification and Characterization |
| 1.1 Overview of Protein Purification and Characterization |
| 1.2 Protein Purification Flow Charts |
| 2 Computational Analysis |
| 2.1 Hydrophobicity Profiles for Protein Sequence Analysis |
| 2.2 Protein Secondary Structure Prediction |
| 2.3 Sequence Similarity Searching Using the BLAST Family of Programs |
| 2.4 Protein Databases on the Internet |
| 2.5 Protein Tertiary Structure Prediction |
| 2.6 Protein Tertiary Structure Modeling |
| 2.7 Comparative Protein Structure Prediction |
| 3 Detection and Assay Methods |
| 3.1 Spectrophotometric Determination of Protein Concentration |
| 3.2 Quantitative Amino Acid Analysis |
| 3.3 In Vitro Radiolabeling of Peptides and Proteins |
| 3.4 Assays for Total Protein |
| 3.5 Biotinylation of Proteins in Solution and on Cell Surfaces |
| 3.6 Metabolic Labeling with Amino Acids |
| 4 Extraction, Stabilization, and Concentration |
| 4.1 Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration |
| 4.2 Selective Precipitation of Proteins |
| 4.3 Long-Term Storage of Proteins |
| 5 Production of Recombinant Proteins |
| 5.1 Production of Recombinant Proteins in Escherichia coli |
| 5.2 Selection of Escherichia coli Expression Systems |
| 5.3 Fermentation and Growth of Escherichia coli for Optimal Protein Production |
| 5.4 Protein Expression in the Baculovirus System |
| 5.5 Culture of Yeast for the Production of Heterologous Proteins |
| 5.6 Overview of Protein Expression by Mammalian Cells |
| 5.7 Production of Recombinant Proteins in Mammalian Cells |
| 5.8 Preparation of Cell Cultures and Vaccinia Virus Stocks |
| 5.9 Generation of Recombinant Vaccinia Viruses |
| 5.10 Choice of Cellular Protein Expression System |
| 5.11 Use of the Gateway System for Protein Expression in Multiple Hosts |
| 6 Purification of Recombinant Proteins |
| 6.1 Overview of the Purification of Recombinant Proteins Produced in Escherichia coli |
| 6.2 Preparation of Soluble Proteins from Escherichia coli |
| 6.3 Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli |
| 6.4 Overview of Protein Folding |
| 6.5 Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli |
| 6.6 Expression and Purification of GST Fusion Proteins |
| 6.7 Expression and Purification of Thioredoxin Fusion Proteins |
| 7 Characterization of Recombinant Proteins |
| 7.1 Overview of the Characterization of Recombinant Proteins |
| 7.2 Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy |
| 7.3 Determining the Identity and Structure of Recombinant Proteins |
| 7.4 Transverse Urea-Gradient Gel Electrophoresis |
| 7.5 Analytical Ultracentrifugation |
| 7.6 Determining the CD Spectrum of a Protein |
| 7.7 Determining the Fluorescence Spectrum of a Protein |
| 7.8 Measuring Protein Thermostability by Differential Scanning Calorimetry |
| 7.9 Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry |
| 8 Conventional Chromatographic Separations |
| 8.1 Overview of Conventional Chromatography |
| 8.2 Ion-Exchange Chromatography |
| 8.3 Gel-Filtration Chromatography |
| 8.4 Hydrophobic-Interaction Chromatography |
| 8.5 HPLC of Peptides and Proteins |
| 9 Affinity Purification |
| 9.1 Lectin Affinity Chromatography |
| 9.2 Dye Affinity Chromatography |
| 9.3 Affinity Purification of Natural Ligands |
| 9.4 Metal-Chelate Affinity Chromatography (MCAC) |
| 9.5 Immunoaffinity Chromatography |
| 9.6 Immunoprecipitation |
| 10 Electrophoresis |
| 10.1 One-Dimensional SDS Gel Electrophoresis of Proteins |
| 10.2 One-Dimensional Electrophoresis Using Nondenaturing Conditions |
| 10.3 Two-Dimensional Gel Electrophoresis |
| 10.4 Protein Detection in Gels Using Fixation |
| 10.5 Electroblotting From Polyacrylamide Gels |
| 10.6 Detection of Proteins on Blot Membranes |
| 10.7 Immunoblot Detection |
| 11 Chemical Analysis |
| 11.1 Enzymatic Digestion of Proteins in Solution |
| 11.2 Enzymatic Digestion of Proteins on PVDF Membranes |
| 11.3 Digestion of Proteins in Gels for Sequence Analysis |
| 11.4 Chemical Cleavage of Proteins in Solution |
| 11.5 Chemical Cleavage of Proteins on Membranes |
| 11.6 Reversed-Phase Isolation of Peptides |
| 11.7 N-Terminal Sequence Analysis |
| 12 Post-Translational Modification: Glycosylation |
| 12.1 Inhibition of N-Linked Glycosylation |
| 12.2 Endoglycosidase and Glycoamidase Release of N-Linked Oligosaccharides |
| 12.3 Detection of Glycophospholipid Anchors on Proteins |
| 13 Post-Translational Modification: Phosphorylation and Phosphatases |
| 13.1 Labeling Cultured Cells with [superscript 32]P[subscript i] and Preparing Cell Lysates for Immunoprecipitation |
| 13.2 Phosphoamino Acid Analysis |
| 13.3 Detection of Phosphorylation by Immunological Techniques |
| 13.4 Detection of Phosphorylation by Enzymatic Techniques |
| 13.5 Permeabilization Strategies to Study Protein Phosphorylation |
| 13.6 Phosphopeptide Mapping and Identification of Phosphorylation Sites |
| 14 Post-Translational Modification: Specialized Applications |
| 14.1 Analysis of Disulfide Bond Formation |
| 14.2 Analysis of Protein Acylation |
| 14.3 Analysis of Protein Prenylation and Carboxyl-Methylation |
| 14.4 Analysis of Oxidative Modification of Proteins |
| 14.5 Analysis of Protein Ubiquitination |
| 15 Chemical Modification of Proteins |
| 15.1 Modification of Cysteine |
| 15.2 Modification of Amino Groups |
| 16 Mass Spectrometry |
| 16.1 Overview of Peptide and Protein Analysis by Mass Spectrometry |
| 16.2 Sample Preparation for MALDI Mass Analysis of Peptides and Proteins |
| 16.3 In-Gel Digestion of Proteins for MALDI-MS Fingerprint Mapping |
| 16.4 Searching Sequence Databases Over the Internet: Protein Identification Using MS-Fit |
| 16.5 Searching Sequence Databases Over the Internet: Protein Identification Using MS-Tag |
| 16.6 Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using a Nanospray Interface |
| 16.7 Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using Microscale Capillary Liquid Chromatography |
| 16.8 Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and SEQUEST Database Matching |
| 16.9 De Novo Peptide Sequencing Via Manual Interpretation of MS/MS Spectra |
| 17 Preparation and Handling of Peptides |
| 17.1 Synthesis of Multiple Peptides on Plastic Pins |
| 17.2 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins |
| 17.3 Synthesis and Application of Peptide Dendrimers as Protein Mimetics |
| 17.4 Disulfide Bond Formation in Peptides |
| 18 Identification of Protein Interactions |
| 18.1 Analysis of Protein-Protein Interactions |
| 18.2 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins |
| 18.3 Phage-Based Expression Cloning to Identify Interacting Proteins |
| 18.4 Detection of Protein-Protein Interactions by Coprecipitation |
| 18.5 High-Throughput Screening for Protein-Protein Interactions Using Yeast Two-Hybrid Arrays |
| 18.6 Identification of Protein Interactions by Far Western Analysis |
| 18.7 Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions |
| 19 Quantitation of Protein Interactions |
| 19.1 Overview of the Quantitation of Protein Interactions |
| 19.2 Titration Calorimetry |
| 19.3 Reduced-Scale Large-Zone Analytical Gel-Filtration Chromatography for Measurement of Protein Association Equilibria |
| 19.4 Size-Exclusion Chromatography with On-Line Light Scattering |
| 20 Peptidases |
| 20.1 Proteases |
| 20.2 Purification and Characterization of Proteasomes from Saccharomyces cerevisiae |
| 20.3 Purification of the Eukaryotic 20S Proteasome |
| 20.4 Serpins (Serine Protease Inhibitors) |
| 20.5 Use of GFP as a Reporter for the Analysis of Sequence-Specific Proteases |
| 20.6 Overexpression and Purification of Active Serine Proteases and Their Variants from Escherichia coli Inclusion Bodies |
| 20.7 Assaying Proteases in Cellular Environments |
| 20.8 Expression, Purification, and Characterization of Caspases |
| 21 Gel-Based Proteome Analysis |
| 21.1 Protein Profiling Using Two-Dimensional Difference Gel Electrophoresis (2-D DIGE) |
| 21.2 Laser Capture Microdissection for Proteome Analysis |
| Appendices |
| A1 Reagents and Solutions |
| A2 Useful Measurements and Data |
| A3 Commonly Used Techniques |
| A4 Selected Suppliers of Reagents and Equipment |
| References |
| Index |
