| Abbreviations |
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vii | |
| Preface |
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ix | |
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Protein purification strategy and equipment |
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1 | (24) |
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1 | (1) |
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Reasons to justify the purification of a protein |
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2 | (1) |
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Loss of protein during a purification schedule |
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2 | (2) |
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Overview of protein structure |
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4 | (1) |
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Properties of proteins that enable purification |
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5 | (2) |
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The range of techniques for protein purification |
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7 | (1) |
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Protein purification strategy |
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7 | (2) |
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The process of protein purification |
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9 | (2) |
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The theory of chromatography |
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11 | (1) |
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Equipment required for protein purification |
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12 | (3) |
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Protein purification chromatographic runs |
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15 | (3) |
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Information required for a protein purification balance sheet |
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18 | (7) |
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25 | (54) |
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25 | (1) |
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25 | (1) |
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26 | (1) |
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Assay to identify a target protein |
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27 | (2) |
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29 | (2) |
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Extraction of protein from cells or tissue |
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31 | (4) |
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Techniques used to disrupt tissue or cells |
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35 | (2) |
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Extraction methods for small amounts of tissue or cells |
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37 | (2) |
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Extraction methods for large amounts of animal/plant tissue or cells |
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39 | (1) |
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Extraction methods for bacterial or yeast cells |
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40 | (1) |
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Points to remember about extraction procedures |
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41 | (1) |
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Techniques for clarifying homogenized extracts (centrifugation) |
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41 | (4) |
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Techniques for clarifying homogenized extracts (aqueous two-phase partitioning) |
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45 | (2) |
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Techniques for concentrating proteins from dilute solutions (laboratory scale) |
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47 | (9) |
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Clarification of process-scale extracts |
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56 | (1) |
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57 | (1) |
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Storage of protein samples |
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58 | (21) |
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Nonaffinity absorption techniques for purifying proteins |
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79 | (18) |
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Ion exchange chromatography (IEX) |
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79 | (5) |
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84 | (2) |
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Hydroxyapatite chromatography |
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86 | (1) |
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Hydrophobic interaction chromatography (HIC) |
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87 | (4) |
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Hydrophobic charge induction chromatography (HCIC) |
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91 | (1) |
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Mixed mode chromatography (MMC) |
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92 | (5) |
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Affinity procedures for purifying proteins |
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97 | (24) |
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97 | (6) |
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103 | (2) |
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Dye ligand affinity chromatography |
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105 | (1) |
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Immobilized metal (ion) affinity chromatography (IMAC) |
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106 | (2) |
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Immunoaffinity chromatography |
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108 | (1) |
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Lectin affinity chromatography |
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109 | (3) |
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Purification of recombinant proteins |
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112 | (1) |
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IMAC for purifying recombinant proteins |
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113 | (1) |
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Fusion proteins for purifying recombinant proteins |
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114 | (2) |
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Affinity partitioning (precipitation) |
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116 | (5) |
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Nonabsorption techniques for purifying proteins |
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121 | (16) |
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Size exclusion chromatography (SEC) |
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121 | (3) |
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Some factors to consider in SEC |
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124 | (3) |
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Preparation and storage of SEC resins |
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127 | (1) |
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127 | (1) |
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SEC to separate protein aggregates or the removal of low amounts of contaminating material |
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127 | (1) |
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Desalting (group separation) |
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128 | (1) |
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SEC in the refolding of denatured proteins |
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128 | (1) |
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Preparative polyacrylamide gel electrophoresis (PAGE) |
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128 | (2) |
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Isolation of proteins from polyacrylamide gels or from nitrocellulose membranes |
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130 | (1) |
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Preparative isoelectric focusing (IEF) |
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131 | (6) |
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Monitoring the purity of protein solutions |
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137 | (24) |
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Electrophoresis of proteins |
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137 | (1) |
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The theory of electrophoresis |
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138 | (1) |
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Polyacrylamide gel electrophoresis (PAGE) |
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139 | (5) |
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144 | (2) |
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Isoelectric focusing (IEF) |
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146 | (1) |
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Capillary electrophoresis (CE) |
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147 | (2) |
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Reversed phase high-pressure liquid chromatography (RP-HPLC) |
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149 | (12) |
| Appendix 1 The common units used in biology |
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161 | (1) |
| Appendix 2 Answers to the exercises |
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162 | (3) |
| Appendix 3 Single-letter code for amino acids |
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165 | (1) |
| Appendix 4 List of suppliers |
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166 | (9) |
| Glossary |
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175 | (10) |
| Index |
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185 | |
