1. Why Purify Enzymes? 1
Arthur Kornberg (with preface by Murray Deutscher)
Section I. Developing Purification Procedures 7
2. Strategies and Considerations for Protein Purifications 9
Stuart Linn
1. General Considerations 10
2. Source of the Protein 15
3. Preparing Extracts 16
4. Bulk or Batch Procedures for Purification 17
5. Refined Procedures for Purification 18
6. Conclusions 19
References 19
3. Use of Bioinformatics in Planning a Protein Purification 21
Richard R. Burgess
1. What You Can Learn from an Amino Acid Sequence 22
2. What You Cannot yet Predict 25
3. Conclusion 26
References 27
4. Preparing a Purification Summary Table 29
Richard R. Burgess
1. Introduction 29
2. The Importance of Footnotes 32
3. The Value of an SDS–Polyacrylamide Gel Analysis on Main
Protein Fractions 32
4. Some Common Mistakes and Problems
Section II. General Methods for Handling
Proteins and Enzymes 35
5. Setting Up a Laboratory 37
Murray P. Deutscher
1. Supporting Materials 38
2. Detection and Assay Requirements 39
3. Fractionation Requirements 40
6. Buffers: Principles and Practice 43
Vincent S. Stoll and John S. Blanchard
1. Introduction 43
2. Theory 44
3. Buffer Selection 45
4. Buffer Preparation 48
5. Volatile Buffers 49
6. Broad-Range Buffers 50
7. Recipes for Buffer Stock Solutions 50
References 56
7. Measurement of Enzyme Activity 57
T. K. Harris and M. M. Keshwani
1. Introduction 58
2. Principles of Catalytic Activity 58
3. Measurement of Enzyme Activity 64
4. Formulation of Reaction Assay Mixtures 69
5. Discussion 71
Acknowledgments 71
References 71
8. Quantitation of Protein 73
James E. Noble and Marc J. A. Bailey
1. Introduction 74
2. General Instructions for Reagent Preparation 75
3. Ultraviolet Absorption Spectroscopy 80
4. Dye-Based Protein Assays 83
5. Coomassie Blue (Bradford) Protein Assay (Range: 1–50 mg) 85
6. Lowry (Alkaline Copper Reduction Assays) (Range: 5–100 mg) 86
7. Bicinchoninic Acid (BCA) (Range: 0.2–50 mg) 88
8. Amine Derivatization (Range: 0.05–25 mg) 89
9. Detergent-Based Fluorescent Detection (Range: 0.02–2 mg) 91
10. General Instructions 91
Acknowledgment 94
References 94
9. Concentration of Proteins and Removal of Solutes 97
David R. H. Evans, Jonathan K. Romero, and Matthew Westoby
1. Chromatography 98
2. Electrophoresis 103
3. Dialysis 104
4. Ultrafiltration 107
5. Lyophilization 113
6. Precipitation 116
7. Crystallization 118
References 118
10. Maintaining Protein Stability 121
Murray P. Deutscher
1. Causes of Protein Inactivation 121
2. General Handling Procedures 122
3. Concentration and Solvent Conditions 122
4. Stability Trials and Storage Conditions 123
5. Proteolysis and Protease Inhibitors 124
6. Loss of Activity 125
Section III. Recombinant Protein
Expression and Purification 129
11. Selecting an Appropriate Method for Expressing
a Recombinant Protein 131
William H. Brondyk
1. Introduction 132
2. Escherichia coli 133
3. Pichia pastoris 135
4. Baculovirus/Insect Cells 136
5. Mammalian Cells 138
6. Protein Characteristics 139
7. Recombinant Protein Applications 143
8. Conclusion 144
References 144
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