文件名称：Protein Biochemistry and Proteomics
文件大小：10.57MB
文件格式：PDF
更新时间：2012-07-08 16:04:39
Protein Biochemistry Proteomics
1 Daily Bread 1 1.1 Making Buffers 1 1.2 Protein Determination 1 1.2.1 BCA Assay 2 1.2.2 Bradford Assay 3 1.2.3 Lowry Assay 3 1.2.4 Starcher Assay 3 1.2.5 Protein Concentration 4 1.3 Gels 4 1.3.1 SDS Gels 5 1.3.2 For the Impatient: SDS Electrophoresis Without Stacker 7 1.3.3 Native Gels 8 1.4 Staining Gels 9 1.4.1 Fixing 9 1.4.2 Staining 9 1.4.3 Drying 12 1.5 Precipitation and Concentration 13 1.5.1 Denaturing Precipitation 13 1.5.2 Native Precipitation 14 1.5.3 Concentration 14 1.6 Blotting 15 1.6.1 Protein Staining on Blots 17 1.6.2 Blocking 18 1.6.3 Immunostaining 19 2+ Binding 20 1.6.4 Ca 1.6.5 Ligand Staining 21 1.7 Autoradiography of Gels and Blots 22 2 Ligand Binding 25 2.1 Radioactive Ligand Marking 26 2.1.1 Iodination of Peptides and Proteins 26 2.1.2 The Day After 28 2.1.3 Iodination of Molecules with Low MW 29 2.1.4 Isolation of Iodized Species 29 2.1.5 Advantages and Disadvantages of Iodination 30 2.1.6 Tritiation 32 2.2 Binding 32 2.2.1 Isolation of Membranes 32 2.2.2 Binding Assay 34 2.2.3 Binding Assays with Membranes 37 2.2.4 Development of Membrane Binding Assays 38 2.2.5 Binding Assays with Soluble Proteins 39 2.2.6 No Binding 55 2.3 Analysis of Binding Data 57 2.3.1 The Binding Reaction in Equilibrium 57 2.3.2 Kinetics 69 vi · Contents 2.4 Cross-linking of Ligands 71 2.4.1 Three-component Cross-linking (3C Cross-linking) 72 2.4.2 Photoaffi nity Cross-linking 77 2.4.3 Controls for Cross-linking Experiments 79 2.5 Purposes 79 3 Solubilization of Membrane Proteins 83 3.1 Detergents 83 3.1.1 Clean Concepts 83 3.1.2 Handling Detergents 85 3.2 Solubilization 87 3.2.1 Solubilization Criteria 92 3.2.2 Physical Parameters of Solubilized Membrane Proteins 93 4 Protein Detection via Functional Measurements 95 4.1 Translocators 95 4.1.1 Liposomes 96 4.1.2 Proteoliposomes 97 4.2 Reconstitution 98 4.2.1 Reconstitution from a Solution 98 4.2.2 Reconstitution in Preformed Liposomes 100 4.3 Flux Assay 100 4.3.1 Infl ux Assay 102 4.3.2 Effl ux Assay 104 4.4 Constructive Thoughts 105 5 Cleaning and Purifying 109 5.1 Pure Fun 109 5.2 Conventional Purifi cation Methods 113 5.2.1 The Column Technique 113 5.2.2 Purifi cation Based on Size Differences 114 5.2.3 Purifi cation Based on Charge Differences 117 5.2.4 Hydrophobic Chromatography 124 5.2.5 The Blue Gel 125 5.3 Affi nity Chromatography 125 5.3.1 Lectin Chromatography 125 5.3.2 Ligand Chromatography 127 5.4 The Purity Test 134 5.5 Profi ting 134 6 Antibodies 137 6.1 Production of Polyclonal Antibodies 140 6.1.1 Antigen 140 6.1.2 Adjuvant 141 6.1.3 Injection and Serum Harvesting 142 6.1.4 Purifi cation of Antibodies 143 6.2 Immunoprecipitation 145 6.2.1 Immunoprecipitation with Immobilized Protein A 145 6.2.2 Immunoprecipitation with Immobilized Antibody 146 6.3 Immunoaffi nity Chromatography 148 6.4 Antibodies Against Unpurifi ed Proteins 148 6.5 Immunological Detection Techniques 150 7 Proteomics 155 7.1 Introduction 155 7.2 Sample Taking 157 Contents · vii 7.3 2D Gel Electrophoresis 161 7.4 Mass Spectroscopy of Peptides and Proteins 165 7.4.1 Mass Spectrometers 165 7.4.2 Sample Preparation for MALDI 170 7.4.3 The Possibilities of MALDI and ESI 172 7.5 Protein Chips 174 7.5.1 Protein Chips with SELDI 174 7.5.2 Fortune Cookies 176 7.5.3 Aptamers 177 7.6 Microsequencing 179 7.6.1 Preparing the Protein 179 7.6.2 Blocked N-Termini 180 7.6.3 Cleaving the Protein into Peptides 181 7.6.4 The Edman Degradation 183 7.6.5 Carboxyterminal Sequencing 183 7.6.6 Ladder Sequencing of Peptides 184 7.7 Strategy 189 8 Subunits 191 8.1 Number and Stoichiometry of Subunits 191 8.1.1 About the Diffi culties with Stoichiometry Determinations 191 8.1.2 N&S with X-ray Structural Analysis 192 8.1.3 N&S with Hybridization Experiments 192 8.1.4 N&S with Cross-linking Experiments 194 8.1.5 N&S with Amino Acid Analyses or Antibodies 199 8.2 What Holds Our World Together? 200 9 Glycoproteins 205 9.1 How, Where, and for What Purpose Are Proteins Glycosylated? 205 9.2 Detecting Glycoproteins in Gels 206 9.3 Detection of Glycoproteins on Blots 206 9.3.1 Nonselective Glycoprotein Stain 206 9.3.2 Selective (Lectin) Stain 208 9.4 Deglycosylation 208 9.4.1 Glycosylation Inhibitor 208 9.4.2 Endoglycosidases 210 9.4.3 Chemical Deglycosylation 214 9.5 The Sugar Chains 216 9.5.1 Monosaccharide Composition 216 9.5.2 Structure and Sequence 216 10 Treasure Island: Writing Papers 225 10.1 Of the Paper 225 10.2 Of Writing a Paper 225 11 Desert Planet: Researching the Literature 227 Last Things 228 Appendix A Professional Resources 229 A.1 Suppliers 229 A.2 Suppliers by Product 229 Index 231