Table of Contents
Last Updated February 2003
-------------------------------------------------------------------------------- Foreword by Leroy E. Hood and Ruedi Aebersold
Preface
Contributors

1 Strategies of Protein Purification and Characterization
2 Computational Analysis
3 Detection and Assay Methods
4 Extraction, Stabilization, and Concentration
5 Production of Recombinant Proteins
6 Purification of Recombinant Proteins
7 Characterization of Recombinant Proteins
8 Conventional Chromatographic Separations
9 Affinity Purification
10 Electrophoresis
11 Chemical Analysis
12 Post-Translational Modification: Glycosylation
13 Post-Translational Modification: Phosphorylation and Phosphatases
14 Post-Translational Modification: Specialized Applications
15 Chemical Modification of Proteins
16 Mass Spectrometry
17 Structural Biology
18 Preparation and Handling of Peptides
19 Identification of Protein Interactions
20 Quantitation of Protein Interactions
21 Peptidases
22 Gel-Based Proteome Analysis (New, December 2002)
Appendices
Indexes

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1 Strategies of Protein Purification and Characterization
1.1 Overview of Protein Purification and Characterization
1.2 Strategies for Protein Purification
1.3 Protein Purification Flow Charts
1.4 Purification of Glutamate Dehydrogenase From Liver and Brain
1.5 Overview of the Physical State of Proteins Within Cells (New, December 2002)

2 Computational Analysis
2.1 Computational Methods for Protein Sequence Analysis
2.2 Hydrophobicity Profiles for Protein Sequence Analysis
2.3 Protein Secondary Structure Prediction
2.4 Internet Basics
2.5 Sequence Similarity Search Using the BLAST Family of Programs
2.6 Protein Databases on the Internet
2.7 Protein Tertiary Structure Prediction
2.8 Protein Tertiary Structure Modeling
2.9 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 Kinetic Assay Methods
3.6 Biotinylation of Proteins in Solution and on Cell Surfaces
3.7 Metabolic Labeling with Amino Acids
3.8 Analysis of Selenocysteine-Containing Proteins
3.9 Solid-Phase Profiling of Proteins

4 Extraction, Stabilization, and Concentration
4.1 Overview of Cell Fractionation
4.2 Purification of Organelles from Mammalian Cells
4.3 Subcellular Fractionation of Tissue Culture Cells
4.4 Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration
4.5 Selective Precipitation of Proteins
4.6 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 Overview of the Baculovirus Expression System
5.5 Protein Expression in the Baculovirus System
5.6 Overview of Protein Expression in Saccharomyces cerevisiae
5.7 Overview of Protein Expression in Pichia pastoris
5.8 Culture of Yeast for the Production of Heterologous Proteins
5.9 Overview of Protein Expression by Mammalian Cells
5.10 Production of Recombinant Proteins in Mammalian Cells
5.11-5.15 The Vaccinia Virus Expression System
5.16 Choice of Cellular Protein Expression System
5.17 Use of the Gateway System for Protein Expression in Multiple Hosts (New, December 2002)

6 Purification of Recombinant Proteins
6.1 Overview of the Purification of Recombinant Proteins Produced in Escherichia coli (Revised, December 2002)
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 Light Scattering
7.9 Measuring Protein Thermostability by Differential Scanning Calorimetry
7.10 Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry
7.11 Rapid Screening of E. coli Extracts by Heteronuclear NMR (New, February 2003)

>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 Chromatofocusing
8.6 Coming soon!
8.7 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
9.5 Immunoaffinity Chromatography
9.6 Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography
9.7 Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems
9.8 Immunoprecipitation

10 Electrophoresis
10.1 One-Dimensional SDS Gel Electrophoresis of Proteins
10.2 One-Dimensional Isoelectric Focusing of Proteins in Slab Gels
10.3 One-Dimensional Electrophoresis Using Nondenaturing Conditions
10.4 Two-Dimensional Gel Electrophoresis
10.5 Protein Detection in Gels Using Fixation
10.6 Protein Detection in Gels Without Fixation
10.7 Electroblotting from Polyacrylamide Gels
10.8 Detection of Proteins on Blot Membranes
10.9 Capillary Electrophoresis of Proteins and Peptides
10.10 Immunoblot Detection
10.11 Autoradiography
10.12 Digital Electrophoresis Analysis
10.13 Capillary Electrophoresis of Peptides and Proteins Using Isoelectric Buffers

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 Removal of N-Terminal Blocking Groups from Proteins
11.8 C-Terminal Sequence Analysis (Revised, February 2003)
11.9 Amino Acid Analysis

12 Post-Translational Modification: Glycosylation
12.1 Overview of Glycoconjugate Analysis
12.2 Metabolic Radiolabeling of Animal Cell Glycoconjugates
12.3 Inhibition of N-Linked Glycosylation
12.4 Endoglycosidase and Glycoamidase Release of N-Linked Oligosaccharides
12.5 Detection of Glycophospholipid Anchors on Proteins
12.6 Determining the Structure of Oligosaccharides N- and O-Linked to Glycoproteins
12.7 Determining the Structure of Glycan Moieties by Mass Spectrometry
12.8 Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine

13 Post-Translational Modification: Phosphorylation and Phosphatases
13.1 Overview of Protein Phosphorylation
13.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
13.3 Phosphoamino Acid Analysis
13.4 Detection of Phosphorylation by Immunological Techniques
13.5 Detection of Phosphorylation by Enzymatic Techniques
13.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
13.7 Assays of Protein Kinases Using Exogenous Substrates
13.8 Permeabilization Strategies to Study Protein Phosphorylation
13.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites
13.10 Use of Protein Phosphatase Inhibitors (New, February 2003)

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 Acids

16 Mass Spectrometry
16.1 Overview of Peptide and Protein Analysis by Mass Spectrometry
16.2 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Analysis of Peptides
16.3 Sample Preparation for MALDI
16.4 MALDI-MS Fingerprint Mapping
16.5 Searching Sequence Databases Over the Internet: MS-Fit
16.6 Searching Sequence Databases Over the Internet: MS-Tag
16.7 Enzymatic Approaches for Obtaining Amino Acid Sequence: On-Target Ladder Sequencing
16.8 ESI-MS Using a Nanospray Interface
16.9 ESI-MS Using Microscale Capillary Liquid Chromatography
16.10 SEQUEST Database Matching
16.11 De Novo Peptide Sequencing Via Manual Interpretation of MS/MS Spectra

17 Structural Biology
17.1 Overview of Protein Structural and Functional Folds
17.2 Overview of Macromolecular Electron Microscopy
17.3 Principles of Macromolecular X-Ray Crystallography
17.4 Crystallization of Soluble Macromolecules
17.5 Introduction to NMR of Proteins
17.6 Probing Protein Structure and Dynamics by Hydrogen Exchange-Mass Spectrometry
17.7 Introduction to Atomic Force Microscopy (AFM) in Biology

18 Preparation and Handling of Peptides
18.1 Introduction to Peptide Synthesis
18.2 Synthesis of Multiple Peptides on Plastic Pins
18.3 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
18.4 Native Chemical Ligation of Polypeptides
18.5 Synthesis and Application of Peptide Dendrimers as Protein Mimetics
18.6 Disulfide Bond Formation in Peptides

19 Identification of Protein Interactions
19.1 Protein-Protein Interactions
19.2 Interaction Trap/Two-Hybrid System
19.3 Phage-Based Expression Cloning to Identify Interacting Proteins
19.4 Detection of Protein-Protein Interactions by Coprecipitation
19.5 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
19.6 High-Throughput Screening for Protein-Protein Interactions Using Yeast Two-Hybrid Arrays
19.7 Identification of Protein Interactions by Far Western Analysis
19.8 Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions

20 Quantitation of Protein Interactions
20.1 Overview of the Quantitation of Protein Interactions
20.2 Measuring Protein Interactions by Optical Biosensors
20.3 Analytical Centrifugation: Equilibrium Approach
20.4 Titration Microcalorimetry
20.5 Large-Zone Analytical Gel-Filtration Chromatography
20.6 Size-Exclusion Chromatography with On-Line Light Scattering
20.7 Analytical Ultracentrifugation: Sedimentation Velocity Analysis (New, February 2003)

21 Peptidases
21.1 Proteases
21.2 Papain-like Cysteine Proteases
21.3 Overview of Pepsin-Like Aspartic Peptidases
21.4 Metalloproteases
21.5 Purification and Characterization of Proteasomes from Saccharomyces cerevisiae
21.6 Purification of the Eukaryotic 20S Proteasome
21.7 Serpins (Serine Protease Inhibitors)
21.8 Caspases
21.9 Use of GFP as a Reporter for the Analysis of Sequence-Specific Proteases
21.10 An Overview of Serine Proteases
21.11 Over-Expression and Purification of Active Serine Proteases and Their Variants From Escherichia coli Inclusion Bodies
21.12 Assaying Proteases in Cellular Environments
21.13 Expression, Purification, and Characterization of Caspases (New, December 2002)

22 Gel-Based Proteome Analysis
22.1 Overview of Proteome Analysis (New, December 2002)
22.2 Protein Profiling Using Two-Dimensional Difference Gel Electrophoresis (2-D DIGE) (New, December 2002)
22.3 Laser Capture Microdissection for Proteome Analysis (New, February 2003)

Appendices
A.1 Useful Data

Characteristics of Amino Acids
A.2 Laboratory Guidelines, Equipment, and Stock Solutions
Laboratory Safety
Safe Use of Radioisotopes
Centrifuges and Rotors
Standard Laboratory Equipment
Commonly Used Reagents
A.3 Commonly Used Techniques
Use of Protein Folding Reagents
Dialysis Suppliers Appendix
Techniques for Mammalian Cell Tissue Culture
Importing Biological Materials
Silanizing Glassware
Protein Precipitation Using Ammonium Sulfate
Statistics: Detecting Differences Among Groups
Analyzing Radioligand Binding Data
A.4 Molecular Biology Techniques
A.5A Theoretical Aspects of the Quantitative Characterization of Ligand Binding

Indexes