r i Protein purification methods, a practical approach 2008 AGI-Information Management Consultants May be used for personal purporses only or by libraries associated to dandelon.com network. I Edited by E L v Harris and S Angal Celltech Ltd, 216 Bath Road, Slough SL1 4EN, UK OIRL PRESS at OXFORD UNIVERSITY PRESS Oxford New York Tokyo
Contents ABBREVIATIONS xv 1. INITIAL PLANNING 1 Introduction E.L.V.Harris 1 What is the protein required for? 1 What source should be used? 2 What is known about the protein? 3 Basic Preparations E.L.V.Harris 4 Equipment 4 Buffers 5 Assays 9 Determination of Total Protein Concentration M.J.Dunn 10 Ultraviolet spectrophotometry 11 Biuret method 12 Lowry procedure 12 Bicinchoninic acid procedure 13 Dye-binding procedure 15 Silver-binding procedure 16 Electrophoretic Analysis Methods M.J.Dunn 18 Properties of polyacrylamide gels 19 Electrophoresis under denaturing conditions 21 Electrophoresis under native conditions 27 Isoelectric focusing 29 Two-dimensional electrophoresis 33 Analysis of electrophoretic profiles 37 Prevention of Uncontrolled Proteolysis R.J.Benyon 40 Proteolytic susceptibility of native proteins 41 Identification of proteolysis as a problem 42 Inhibition of proteases 49 Removal of proteases 50 Purification Strategy E.L.V.Harris 51 Preserving activity 55 Planning a purification strategy 57 Troubleshooting 64 Acknowledgements 64 64 2. CLARIFICATION AND EXTRACTION 67 Introduction E. L.V.Harris 67 Clarification P. N.Whittington 69 Centrifugation 71 IX
Flocculation Microfiltration Disruption T. Salusbury Pre-treatment Factors affecting ease of disruption Consequences of disruption Methods of tissue and cell disruption Isolation of Organelles Isolation of nuclei G.H.Goodwin Isolation of microsomes N.Lambert Isolation of mitochondria and mitochondrial enzymes D.Griffiths Isolation of chloroplasts M.R.Hartley Acknowledgements CONCENTRATION OF THE EXTRACT Introduction E. L. V. Harris Addition of a Dry Matrix Polymer Ultrafiltration Equipment Operation Other applications of ultrafiltration Freeze-drying or Lyophilization Removal of Salts and Exchange of Buffer Dialysis Diafiltration Gel filtration Purification and Concentration by Precipitation Precipitation by alteration of the ph Precipitation by decreasing the ionic strength Precipitation by increasing the ionic strength (salting-out) Precipitation by organic solvents Precipitation by organic polymers Precipitation by denaturation Aqueous Two-phase Partitioning B.A.Andrews and J.A.Asenjo Equipment and materials Optimization Using phase partitioning in a purification procedure Affinity partitioning Acknowledgements 75 80 87 87 88 89 90 97 97 101 108 114 121 122 125 125 125 126 131 139 147 148 149 149 151 151 151 152 154 154 157 160 160 161 164 166 170 172 172 172
4. SEPARATION BASED ON STRUCTURE 175 S.Roe Introduction 175 General Concepts 175 Chromatography 175 Adsorption/desorption 175 Compatibility 175 Capacity 176 Selectivity 176 Resolution 177 The efficiency of a separation 177 Cost 179 Matrix Materials 179 Cellulose 181 Agarose 181 Dextran 182 Polyacrylamide 183 Natural earths 184 Silica 184 Porous glass 184 Polystyrene and phenol-formaldehyde 184 Chromatographic Equipment and Basic Procedures 186 Low pressure equipment 187 Basic procedures in low pressure chromatography 189 Medium and high pressure equipment 197 Basic procedures in HPLC/MPLC 199 Separation on the Basis of Charge 200 Theory of ion exchange 201 Selection of conditions for ion exchange purifications 203 Procedures in ion exchange separations 211 High performance ion exchange chromatography 215 Separation using chromatofocusing 216 Purification Based on Hydrophobicity 221 Theory of hydrophobic interaction chromatography 222 Experimental conditions for low pressure HIC 225 High performance HIC 228 Advantages and disadvantages of HIC 228 Reverse-phase chromatography 230 Advantages and disadvantages of reverse-phase chromatography 232 Metal Chelate Chromatography 232 Theory of metal chelate chromatography 233 Experimental conditions 234 Covalent Chromatography 235 Experimental techniques 235 Advantages and disadvantages 237 xi
Other Adsorption Techniques Hydroxylapatite High performance hydroxylapatite 5. PURIFICATION BY EXPLOITATION OF ACTIVITY Introduction S.Angal and P.D.G.Dean Design and Preparation of Affinity Adsorbents S.Angal and P.D.G.Dean Choice of ligand Selection of the matrix Choice of spacer Activation and coupling chemistry Estimation of ligand concentration Use of Affinity Adsorbents S.Angal and P.D.G.Dean Initial questions Selection of conditions for operation Estimation of capacity Ligand efficiency Application to protein purification Preparation of Adsorbents Using Oxirane Acrylic Beads and Their Use M.Cusack and R.J.Beynon Properties of Eupergit C Use of Eupergit C Conclusions Lectin Affinity Chromatography C.Sutton Materials for lectin chromatography Procedures for using immobilized lectins Immunopurification C.R.Hill, L.G.Thompson and A.C.Kenney Antibody selection Immobilization of antibodies on CNBr-activated Sepharose Procedures for immunopurification Acknowledgements 6. SEPARATION ON THE BASIS OF SIZE: GEL PERMEATION CHROMATOGRAPHY A.Z.Preneta xii Introduction Gel Permeation Media Choice of gel matrix Experimental Equipment The column
Preparation of the gels 299 Packing the column 299 Choice of eluent 300 Sample size, composition and application 300 Chromatography 301 Column cleaning and storage 304 HPLC and FPLC 304 Applications of GPC 305 Determination of molecular weights 305 305 APPENDIX I Suppliers 307 APPENDIX II Useful recipes 311 INDEX 313 xin