Current Protocols in Protein Science
Table of Contents

Foreword by Leroy E. Hood and Ruedi Aebersold
Preface
Contributors 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

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 Revised, August 2003)
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 Revised, August 2003)
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 Revised, May 2003
4.4 Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration
4.5 Selective Precipitation of Proteins
4.6 Long-Term Storage of Proteins
4.7 Extraction of Proteins from Plant Tissues  

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

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 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 

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
8.8 Expanded-Bed Adsorption Chromatography

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
Unit 9.9 Overview of Affinity Tags for Protein Purification  

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 
11.9 Amino Acid Analysis
11.10 N-Terminal Sequence Analysis of Proteins and Peptides
11.11 Determination of Disulfide-Bond Linkages in Proteins 

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 ³²P_i 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 

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
14.6 Analysis of Protein S-Nitrosylation  
14.7 Tyrosine O-Sulfation    

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 
17.8 Raman Spectroscopy of Proteins (New, August 2003) 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
18.7 Guide for Resin and Linker Selection in Solid-Phase Peptide Synthesis

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
19.9 Identifying Protein Interactions by Hydroxyl-Radical Protein Footprinting  

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 

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 
21.14 Expression, Purification, and Characterization of Aspartic Endopeptidases: <I>Plasmodium Plasmepsins and Short Recombinant Human Pseudocathepsin D (New, May 2003)
21.15 Zymography of Metalloproteinases
21.16 Monitoring Metalloproteinase Activity Using Synthetic Fluorogenic Substrates
21.17 Applications for Chemical Probes of Proteolytic Activity

22 Gel-Based Proteome Analysis

22.1 Overview of Proteome Analysis
22.2 Protein Profiling Using Two-Dimensional Difference Gel Electrophoresis (2-D DIGE) 
22.3 Laser Capture Microdissection for Proteome Analysis 
22.4 Preparing Protein Extracts for Quantitative Two-Dimensional Gel Comparison
22.5 Isolation of Organelles and Prefractionation of Protein Extracts Using Free-Flow Electrophoresis  

23 Non-Gel-Based Proteome Analysis
Introduction
23.1 Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry 
 23.2 Quantitative Protein Profile Comparisons Using the Isotope-Coded Affinity Tag Method
23.3 Proteomic Analysis Using 2-D Liquid Separations of Intact Proteins From Whole-Cell Lysates 
23.4 Quantitative Protein Analysis Using Proteolytic [¹⁸O]Water Labeling   
23.5 Protein Microarrays 

Appendices

Appendix 1 Useful Data
1B Commonly Used Detergents 
1C Conversion Factors and Half-Life Information for Radioactivity  )
1D Common Conversion Factors   

Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions      
2A Laboratory Safety     
2B Safe Use of Radioisotopes 
2C Centrifuges and Rotors  
2D Standard Laboratory Equipment
2E Commonly Used Reagents

Appendix 3 Commonly Used Techniques      
3A Use of Protein Folding Reagents 
3B Dialysis      
3C Techniques for Mammalian Cell Tissue Culture  
3D Importing Biological Materials    
3E Silanizing Glassware   
3F Protein Precipitation Using Ammonium Sulfate  
3G Statistics: Detecting Differences Among Groups 
3H Analyzing Radioligand Binding Data     

Appendix 4 Molecular Biology Techniques     
4A Media Preparation and Bacteriological Tools     
4B Growth in Liquid or Solid Media
4C Preparation of Plasmid DNA      
4D Introduction of Plasmid DNA into Cells
4E Purification and Concentration of DNA from Aqueous Solutions    
4F Agarose Gel Electrophoresis

4G Southern Blotting     
4H Hybridization Analysis of DNA Blots    
4I Digestion of DNA with Restriction Endonucleases   
4J The Polymerase Chain Reactionps
4K Quantitation of Nucleic Acids with Absorption Spectroscopy     
4L Growth and Manipulation of Yeast     

Appendix 5 Biophysical Methods: Data Analysis      
5A Theoretical Aspects of the Quantitative Characterization of Ligand Binding     

SUPPLIERS APPENDIX       Selected Suppliers of Reagents and Equipment