A GUIDE TO PROTEIN ISOLATION

Transcription

1 A GUIDE TO PROTEIN ISOLATION

2 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

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4 Contents Acknowledgements... Preface... Chapter1 An overview of protein isolation WHY DO IT? PROPERTIES OF PROTEINS THE CONCEPTUAL BASIS OF PROTEIN ISOLATION Where to start? When to stop? THE PURIFICATION TABLE CHAPTER 1 STUDY QUESTIONS... 7 Chapter 2 Assay, extraction and sub-cellular fractionation BUFFERS Making a buffer Buffers of constant ionic strength ASSAYS FOR ACTIVITY Enzyme assays The progress curve The enzyme dilution curve The substrate dilution curve The effect of ph on enzyme activity The effect of temperature on enzyme activity ASSAY FOR PROTEIN CONTENT Absorption of ultraviolet light ix xi

5 vi Contents The biuret assay The Lowry assay The bicinchoninic acid assay The Bradford assay METHODS FOR EXTRACTION OF PROTEINS Osmotic shock Pestle homogenisers The Waring blendor and Virtis homogeniser The Polytron/Ultra-Turrax-typehomogeniser Grinding The Parr bomb Extrusion under high pressure Sonication Enzymic digestion CLARIFICATION OF THE EXTRACT CENTRIFUGAL SUB-CELLULAR FRACTIONATION Density gradient centrifugation CHAPTER 2 STUDY QUESTIONS Chapter 3 Concentration of the extract FREEZE DRYING Theoretical and practical considerations in freeze-drying Some tips on vacuum DIALYSIS The Donnan membrane effect Counter-current dialysis Concentration by dialysis (concentrative dialysis) Perevaporation ULTRAFILTRATION Desalting or buffer exchange by ultrafiltration Size fractionation by ultrafiltration CONCENTRATION/FRACTIONATION BY SALTING OUT Why ammonium sulfate? Empirical observations Three-phase partitioning (TPP) FRACTIONAL PRECIPITATION WITH POLYETHYLENE GLYCOL PRECIPITATION WITH ORGANIC SOLVENTS DYE PRECIPITATION CHAPTER 3 STUDY QUESTIONS... 70

6 Contents Chapter 4 Cromatography PRINCIPLES OF CHROMATOGRAPHY The effect of particle size The effect of the mobile phase flow rate Linear and volumetric flow rates EQUIPMENT FOR LOW PRESSURE LIQUID CHROMATOGRAPHY The column Moving the mobile phase Monitoring the effluent and collecting fractions Refrigeration ION-EXCHANGE CHROMATOGRAPHY (IEC) Ion-exchange resins Gradient generators Choosing the ph An ion-exchange chromatography run CHROMATOFOCUSING MOLECULAR EXCLUSION CHROMATOGRAPHY (MEC) The effect of gel sphere size on V The manufacture of small, uniform, gel spheres Determination of MW by MEC Gels used in MEC An MEC run HYDROXYAPATITE CHROMATOGRAPHY The mechanism of hydroxyapatite chromatography AFFINITY CHROMATOGRAPHY HYDROPHOBIC INTERACTION (HI) CHROMATOGRAPHY CHAPTER 4 STUDY QUESTIONs PRINCIPLES OF ELECTROPHORESIS The effect of the buffer BOUNDARY (TISELIUS) ELECTROPHORESIS PAPER ELECTROPHORESIS Electroendosmosis CELLULOSE ACETATE MEMBRANE ELECTROPHORESIS AGAROSE GEL ELECTROPHORESIS STARCH GEL ELECTROPHORESIS POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) Disc electrophoresis Isotachophoresis SDS-PAGE An SDS-PAGE zymogram for proteinases PORE GRADIENT GEL ELECTROPHORESIS vii

7 viii Contents 5.10 ISOELECTRIC FOCUSING Establishing a ph gradient Control of convection Applying the sample and measuring the ph gradient An analytical IEF system Preparative IEF D ELECTROPHORESIS NON-LINEAR ELECTROPHORESIS CHAPTER 5 STUDY QUESTIONS Chapter 6 Immunological methods THE STRUCTURE OF ANTIBODIES ANTIBODY PRODUCTION Making an antiserum IMMUNOPRECIPITATION Immuno single diffusion Mancini radial diffusion Immuno double diffusion Ouchterlony double diffusion analysis Determination of diffusion coefficients IMMUNOELECTROPHORESIS Cross-over electrophoresis Rocket electrophoresis Grabar-Williams immunoelectrophoresis Clarke-Freeman 2-D immunoelectrophoresis AMPLIFICATION METHODS Complement fixation Radioimmunoassay (RIA) Enzyme amplification Enzyme linked immunosorbent assay (ELISA) Immunoblotting Immunogold labeling with silver amplification Colloid agglutination CHAPTER 6 STUDY QUESTIONS INDEX

8 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

9 xi Preface It is a truism of science that the more fundamental the subject, the more universally applicable it is. Nevertheless, it is important to strike a level of fundamentalness appropriate to the task in hand. For example, an in-depth study of the mechanics of motor cars would tell one nothing about the dynamics of traffic. Traffic exists on a different level - it is dependent upon the existence of motor vehicles but the physics and mathematics of traffic can be adequately addressed by considering motor vehicles as mobile blobs, with no consideration of how they become mobile. To start a discourse on traffic with a consideration of the mechanics of motor vehicles would thus be inappropropriate. In writing this volume, I have wrestled with the question of the appropriate level at which to address the physics underlying many of the techniques used in protein isolation. I have tried to strike a level as would be used by a mechanic (with perhaps a slight leaning towards an engineer) - i.e. a practical level, offering appropriate insight but with minimal mathematics. Some people involved in biochemical research have a minimal grounding in chemistry and physics and so I have tried to keep it as simple as possible. Besides trying to find the right level, I have tried to show that the physical principles which can be employed in protein isolation are, in fact, ubiquitously applicable principles with which students may be well familiar, though perhaps in different contexts. These ubiquitously applicable principles - once identified as such - turn out to be old and familiar friends, with whom one can have a great deal of fun when applied to the challenges of protein isolation.

10 xii Preface In an uncertain world one never knows what the future will bring - who knows whether the economy, the state of world politics, or the weather, will be better or worse this time next year than it is now? - but one of the enduring attractions of science is that, because of the labours of scientists thoughout the world, it is almost certain that, this time next year we ll have greater understanding and insight. This book is offered in the spirit of sharing some of the insights that I have gained in my career in Biochemistry. In some instances, I might have got hold of the wrong end of the stick. Where this is the case, I would welcome comment so that we might all learn - as we always do - from the errors. Clive Dennison