A Guide to Protein Isolation

AGUIDETO PROTEIN ISOLATION This page intentionally left blank. (cid:2)(cid:3)(cid:4)(cid:5)(cid:1) (cid:6)(cid:7)(cid:8)(cid:9)(cid:1) (cid:4)(cid:10)(cid:11)(cid:9)(cid:10)(cid:11)(cid:4)(cid:12)(cid:10)(cid:7)(cid:13)(cid:13)(cid:14)(cid:1) (cid:13)(cid:9)(cid:15)(cid:11)(cid:1) (cid:16)(cid:13)(cid:7)(cid:10)(cid:17)(cid:18) A GUIDE TO PROTEIN ISOLATION by Clive Dennison School of Molecular mid Cellular Biosciences, University of Natal, Pietermaritzburg. South Africa KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW eBook ISBN: 0-306-46868-9 Print ISBN: 0-792-35751-5 ©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: http://www.kluweronline.com and Kluwer's eBookstore at: http://www.ebooks.kluweronline.com Contents Acknowledgements........................................................ ix Preface............................................................................ xi Chapter1 An overview of protein isolation .............................. 1 1.1 WHYDOIT?..................................................................................... 1 1.2 PROPERTIES OF PROTEINS................................................................ 2 1.3 THE CONCEPTUAL BASIS OF PROTEIN ISOLATION ............................ 3 1.3.1 Wheretostart?....................................................................... 4 1.3.2Whentostop?..................................................................... 5 1.4 THE PURIFICATION TABLE ............................................................... 6 1.5 CHAPTER 1 STUDY QUESTIONS ........................................................ 7 Chapter 2 Assay, extraction and sub-cellular fractionation.... 8 2.1 BUFFERS .......................................................................................... 8 2.1.1 Makingabuffer....................................................................... 11 2.1.2Buffers ofconstantionicstrength........................................... 13 2.2 ASSAYS FOR ACTIVITY..................................................................... 15 2.2.1 Enzyme assays ......................................................................... 16 2.2.1.1 The progress curve ........................................................... 16 2.2.1.2 The enzyme dilution curve .............................................. 17 2.2.1.3 The substrate dilution curve ............................................ 18 2.2.1.4 The effect of pH on enzyme activity ............................... 19 2.2.1.5Theeffectoftemperatureonenzymeactivity............. 21 2.3 ASSAY FOR PROTEIN CONTENT ........................................................ 21 2.3.1 Absorption of ultraviolet light ................................................. 22 vi Contents 2.3.2 The biuret assay ...................................................... 23 2.3.3 The Lowry assay .......... ..................................................... 23 2.3.4 The bicinchoninic acid assay.............................................. 24 2.3.5 The Bradford assay......................................................... 24 2.4 METHODS FOR EXTRACTION OF PROTEINS ............................. 24 2.4.1 Osmotic shock ......................................................... 25 2.4.2Pestlehomogenisers ........................................................ 26 2.4.3TheWaringblendorandVirtishomogeniser .................... 27 2.4.4ThePolytron/Ultra-Turrax-typehomogeniser .................. 28 2.4.5 Grinding ............................................................................... 28 2.4.6TheParrbomb ................................................................... 29 2.4.7 Extrusion under high pressure......................................... 29 2.4.8 Sonication............................................................................. 30 2.4.9 Enzymic digestion ................................................................. 30 2.5 CLARIFICATION OF THE EXTRACT ................................................. 31 2.6 CENTRIFUGAL SUB-CELLULAR FRACTIONATION ................................... 31 2.6.1 Density gradient centrifugation ......................................... 36 2.7 CHAPTER 2 STUDY QUESTIONS ................................................. 40 Chapter 3 Concentration of the extract ........................................... 41 3.1 FREEZE DRYING ................................................................................ 41 3.1.1Theoretical and practical considerations in freeze-drying .. 42 3.1.2 Some tips on vacuum ............................................................ 46 3.2 DIALYSIS.......................................................................................... 48 3.2.1 The Donnan membrane effect ........................................... 50 3.2.2 Counter-current dialysis ......................................................... 51 3.2.3Concentration by dialysis (concentrative dialysis) ............. 52 3.2.4 Perevaporation ......................................................................... 52 3.3 ULTRAFILTRATION........................................................................ 53 3.3.1Desalting or buffer exchange by ultrafiltration .................. 56 3.3.2 Size fractionation by ultrafiltration ......................................... 56 3.4 CONCENTRATION/FRACTIONATION BY SALTING OUT ....................... 57 3.4.1Whyammoniumsulfate?................................................... 57 3.4.2Empirical observations....................................................... 60 3.4.3 Three-phase partitioning (TPP) .......................................... 64 3.5 FRACTIONAL PRECIPITATION WITH POLYETHYLENE GLYCOL............ 67 3.6 PRECIPITATION WITH ORGANIC SOLVENTS ............................................ 67 3.7 DYE PRECIPITATION ........................................................................... 68 3.8 CHAPTER 3 STUDY QUESTIONS ............................................................ 70 Contents vii Chapter 4 Cromatography....................................................... 71 4.1 PRINCIPLES OF CHROMATOGRAPHY ..................................................... 71 4.1.1 The effect of particle size ........................................................ 76 4.1.2 The effect of the mobile phase flow rate ............................ 78 4.1.2.1 Linear and volumetric flow rates. .................................. 79 4.2 EQUIPMENT FOR LOW PRESSURE LIQUID CHROMATOGRAPHY ........... 80 4.2.1 The column ......................................................................... 80 4.2.2 Moving the mobile phase ........................................................ 82 4.2.3 Monitoring the effluent and collecting fractions................. 85 4.2.4Refrigeration ....................................................................... 86 4.3 ION-EXCHANGE CHROMATOGRAPHY (IEC) ........................................ 87 4.3.1 Ion-exchange “resins” ......................................................... 89 4.3.2 Gradient generators ....................................................................... 92 4.3.3 Choosing the pH ............................................................................ 94 4.3.4 An ion-exchange chromatography run ................................ 95 4.4 CHROMATOFOCUSING............................................................................... 97 4.5 MOLECULAR EXCLUSION CHROMATOGRAPHY (MEC) ........................ 97 4.5.1 The effect of gel sphere size on V ...................................... 100 0 4.5.2 The manufacture of small, uniform, gel spheres ................ 102 4.5.3 Determination of MW by MEC........................................... 102 4.5.4 Gels used in MEC ................................................................... 104 4.5.5 An MEC run ............................................................................ 108 4.6 HYDROXYAPATITE CHROMATOGRAPHY .......................................... 108 4.6.1 The mechanism of hydroxyapatite chromatography........... 109 4.7 AFFINITY CHROMATOGRAPHY ........................................................ 110 4.8 HYDROPHOBIC INTERACTION (HI) CHROMATOGRAPHY ..................... 111 4.9 CHAPTER 4 STUDY QUESTIONs ....................................................... 112 5.1PRINCIPLES OF ELECTROPHORESIS ....................................................... 115 5.1.1 The effect of the buffer ........................................................... 119 5.2 BOUNDARY (TISELIUS) ELECTROPHORESIS....................................... 122 5.3 PAPER ELECTROPHORESIS ................................................................... 123 5.3.1 Electroendosmosis .................................................................. 124 5.4 CELLULOSE ACETATE MEMBRANE ELECTROPHORESIS .................... 125 5.5 AGAROSE GEL ELECTROPHORESIS ...................................................... 126 5.6 STARCH GEL ELECTROPHORESIS ........................................................ 127 5.7 POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) ......................... 129 5.7.1 Disc electrophoresis .............................................................. 129 5.7.1.1 Isotachophoresis ................................................................. 132 5.8 SDS-PAGE.................................................................................... 133 5.8.1 An SDS-PAGE zymogram for proteinases .......................... 135 5.9 PORE GRADIENT GEL ELECTROPHORESIS ........................................ 135 viii Contents 5.10 ISOELECTRIC FOCUSING ................................................................... 136 5.10.1 Establishing a pH gradient .................................................... 137 5.10.2Controlofconvection............................................................ 140 5.10.3 ApplyingthesampleandmeasuringthepHgradient....... 140 5.10.3.1 AnanalyticalIEFsystem............................................... 140 5.10.3.2PreparativeIEF.............................................................. 142 5.11 2-D ELECTROPHORESIS .................................................................. 143 5.12 NON-LINEAR ELECTROPHORESIS .................................................... 143 5.13 CHAPTER 5 STUDY QUESTIONS ....................................................... 148 Chapter 6 Immunological methods ...................................................... 150 6.1 THE STRUCTURE OF ANTIBODIES ...................................................... 150 6.2 ANTIBODY PRODUCTION................................................................ 151 6.2.1 Making an antiserum................................................................ 154 6.3 IMMUNOPRECIPITATION................................................................. 156 6.3.1 Immunosinglediffusion........................................................... 158 6.3.1.1 Manciniradialdiffusion................................................... 159 6.3.2Immunodoublediffusion........................................................ 160 6.3.2.1 Ouchterlonydoublediffusionanalysis............................. 161 6.3.2.2Determinationofdiffusion coefficients .......................... 162 6.4 IMMUNOELECTROPHORESIS ........................................................... 164 6.4.1 Cross-overelectrophoresis....................................................... 164 6.4.2Rocketelectrophoresis............................................................. 165 6.4.3Grabar-Williamsimmunoelectrophoresis................................ 165 6.4.4Clarke-Freeman2-Dimmunoelectrophoresis .......................... 166 6.5 AMPLIFICATION METHODS ................................................................ 168 6.5.1 Complementfixation............................................................... 168 6.5.2 Radioimmunoassay (RIA)....................................................... 170 6.5.3Enzymeamplification............................................................... 171 6.5.3.1Enzymelinkedimmunosorbentassay(ELISA)............ 171 6.5.3.2Immunoblotting................................................................ 173 6.5.4Immunogoldlabelingwithsilveramplification....................... 175 6.5.5 Colloidagglutination................................................................ 176 6.6 CHAPTER 6 STUDY QUESTIONS ...................................................... 179 INDEX...................................................................................................... 181 Acknowledgements Some of the credit for this book should go to my mentors, from whom I first received the “baton” of science and an introduction to proteins, especially Drs George Quicke, Leon Visser, Ivor Dreosti, John Brand and Dennis Luck. I am equally indebted to the students to whom I subsequently passed on the “baton” who, by their searching questions, have contributed significantly to my education and thus to the contents of this book, especially Drs Bill Lindner, Robert Pike, Theresa Coetzer, Edith Elliott, Phil Fortgens and Frieda Dehrmann and the many others who over the years endured my Techniques course. Drs Elliott and Dehrmann also provided a valuable critique of the manuscript. Other scientific collaborators and friends who have offered invaluable encouragement at various stages of my career are Drs Irv Liener and Rex Lovrien, of University of Minnesota, St Paul, Dr Bonnie Sloane of Wayne State University, Detroit, Dr Jim Travis, of University of Georgia, Athens, Dr Vito Turk, Jozef Stefan Institute, Ljubljana, and Dr Ken Scott of Auckland University. Dr Gareth Griffiths, of the EMBL, Heidelberg, has also been a special friend to both my students and myself. With hindsight I can see that the scientific imperative of objectivity - of removing the man from the experiments - when it becomes a habit of life, may tend to remove the humanity from the man. I apologise to those near and dear to me who have suffered as a consequence. ix