Protein analysis and purification : benchtop techniques

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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. LysisPreparation 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 Filtration Chromatography

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