Dennison С. A Guide to Protein Isolation

GUIDE

PROTEIN

ISOLATION

    

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A GUIDE TO PROTEIN ISOLATION

Clive Dennison

University of Natal,

South Africa

School of Molecular mid Cellular Biosciences,

Pietermaritzburg.

KLUWER ACADEMIC PUBLISHERS

NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: 0-306-46868-9

Print ISBN: 0-792-35751-5

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Contents

Acknowledgements........................................................ ix

Preface............................................................................ xi

Chapter

An overview of protein isolation .............................. 1

1.1 W

HY DO IT? ..................................................................................... 1

1.2 P

ROPERTIES OF PROTEINS ................................................................ 2

1.3

CONCEPTUAL BASIS OF PROTEIN ISOLATION ............................ 3

1.3.1 Where to start? ....................................................................... 4

1.4 T

HE PURIFICATION TABLE ............................................................... 6

1.3.2 When to stop?..................................................................... 5

1.5 C

HAPTER

STUDY QUESTIONS ........................................................ 7

Chapter 2

Assay, extraction and sub

cellular fractionation .... 8

2.1 BUFFERS .......................................................................................... 8

2.1.1 Making a buffer.......................................................................

2.1.2 Buffers of constant ionic strength ........................................... 13

2.2 A

SSAYS 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 ............................................

2.2.1.4 The effect of pH on enzyme activity ............................... 19

2.2.1.5 The effect of temperature on enzyme activity ............. 21

2.3.1 Absorption of ultraviolet light ................................................. 22

2.3 A

SSAY FOR PROTEIN CONTENT ........................................................ 21

vi Contents

2.3.2 The biuret assay ...................................................... 23

2.3.4 The bicinchoninic acid assay .............................................. 24

2.3.3 The Lowry assay ..........

2.3.5 The Bradford assay ......................................................... 24

2.4.1 Osmotic shock ......................................................... 25

2.4.2 Pestle homogenisers ........................................................ 26

2.4.3 The Waring blendor and Virtis homogeniser .................... 27

2.4.4 The Polytron/Ultra

Turrax

typehomogeniser .................. 28

2.4.5 Grinding ............................................................................... 28

2.4.6 The Parr bomb ................................................................... 29

2.4.7 Extrusion under high pressure ......................................... 29

2.4.8 Sonication ............................................................................. 30

2.4.9 Enzymic digestion ................................................................. 30

2.6 C

ENTRIFUGAL SUB-CELLULAR FRACTIONATION

................................... 31

2.6.1 Density gradient centrifugation ......................................... 36

2.7 C

HAPTER 2 STUDY QUESTIONS ................................................. 40

..................................................... 23

2.4 METHODS FOR EXTRACTION OF PROTEINS ............................. 24

2.5 C

LARIFICATION OF THE EXTRACT ................................................. 31

Chapter 3

Concentration of the extract

........................................... 41

3.1 F

REEZE DRYING ................................................................................ 41

3.1.1 Theoretical and practical considerations in freeze

drying .. 42

3.1.2 Some tips on vacuum ............................................................ 46

3.2 D

IALYSIS .......................................................................................... 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 U

LTRAFILTRATIO

N ........................................................................ 53

3.3.1 Desalting or buffer exchange by ultrafiltration .................. 56

3.3.2 Size fractionation by ultrafiltration ......................................... 56

3.4.1 Why ammonium sulfate? ................................................... 57

3.4.2 Empirical observations ....................................................... 60

3.4.3 Three

phase partitioning (TPP) .......................................... 64

3.4 C

ONCENTRATION/FRACTIONATION BY SALTING OUT ....................... 57

3.5 F

RACTIONAL PRECIPITATION WITH POLYETHYLENE GLYCOL ............ 67

3.6 P

RECIPITATION WITH ORGANIC SOLVENTS ............................................ 67

3.7 D

YE PRECIPITATION ........................................................................... 68

3.8 C

HAPTER 3 STUDY QUESTIONS ............................................................ 70

Contents vii

Chapter 4

Cromatography.......................................................

4.1 P

RINCIPLES 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 E

QUIPMENT 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

4.3.1 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

4.2.4 Refrigeration ....................................................................... 86

4.3 I

ON-EXCHANGE CHROMATOGRAPHY (IEC) ........................................ 87

4.4 C

HROMATOFOCUSING ............................................................................... 97

4.5.1 The effect of gel sphere size on V

0 ...................................... 100

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 H

YDROXYAPATITE CHROMATOGRAPHY .......................................... 108

4.6.1 The mechanism of hydroxyapatite chromatography........... 109

4.7 A

FFINITY CHROMATOGRAPHY ........................................................ 110

4.8 H

YDROPHOBIC INTERACTION (HI) CHROMATOGRAPHY ..................... 111

4.9 CHAPTER 4 STUDY QUESTIONs ....................................................... 112

5.1

RINCIPLES OF ELECTROPHORESIS ....................................................... 115

5.1.1 The effect of the buffer ........................................................... 119

5.2 B

OUNDARY (TISELIUS) ELECTROPHORESIS....................................... 122

5.3 P

APER ELECTROPHORESIS ................................................................... 123

5.3.1 Electroendosmosis .................................................................. 124

4.5 M

OLECULAR EXCLUSION CHROMATOGRAPHY (MEC) ........................ 97

5.4 C

ELLULOSE ACETATE MEMBRANE ELECTROPHORESIS .................... 125

5.5 A

GAROSE GEL ELECTROPHORESIS ...................................................... 126

5.6 S

TARCH GEL ELECTROPHORESIS ........................................................ 127

5.7 P

OLYACRYLAMIDE 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 P

ORE GRADIENT GEL ELECTROPHORESIS ........................................ 135

viii Contents

5.10 I

SOELECTRIC FOCUSING ................................................................... 136

5.10.3 Applying the sample and measuring the pH gradient ....... 140

5.10.3.1 An analytical IEF system ............................................... 140

5.10.3.2 Preparative IEF .............................................................. 142

5.12 N

ON-LINEAR ELECTROPHORESIS .................................................... 143

5.13 CHAPTER 5 STUDY QUESTIONS ....................................................... 148

5.10.1 Establishing a pH gradient .................................................... 137

5.10.2 Control of convection............................................................ 140

5.11 2

DELECTROPHORESIS .................................................................. 143

Chapter 6

Immunological methods ...................................................... 150

6.1 THE STRUCTURE OF ANTIBODIES ...................................................... 150

6.2 ANTIBODY PRODUCTION ................................................................ 151

6.3 I

MMUNOPRECIPITATION ................................................................. 156

6.3.1 Immuno single diffusion........................................................... 158

6.3.2 Immuno double diffusion ........................................................ 160

6.3.2.1 Ouchterlony double diffusion analysis............................. 161

6.3.2.2 Determination of diffusion coefficients .......................... 162

6.4 I

MMUNOELECTROPHORESIS ........................................................... 164

6.4.1 Cross

over electrophoresis ....................................................... 164

6.4.3 Grabar

Williams immunoelectrophoresis ................................ 165

6.4.4 Clarke

Freeman 2

D immunoelectrophoresis .......................... 166

6.5 AMPLIFICATION METHODS ................................................................ 168

6.2.1 Making an antiserum................................................................ 154

6.3.1.1 Mancini radial diffusion ................................................... 159

6.4.2 Rocket electrophoresis ............................................................. 165

6.5.1 Complement fixation............................................................... 168

6.5.2 Radioimmunoassay (RIA)....................................................... 170

6.5.3 Enzyme amplification............................................................... 171

6.5.3.1 Enzyme linked immunosorbent assay (ELISA)............ 171

6.5.3.2 Immunoblotting ................................................................ 173

6.5.4 Immunogold labeling with silver amplification ....................... 175

6.5.5 Colloid agglutination................................................................ 176

6.6 C

HAPTER 6 STUDY QUESTIONS ...................................................... 179

NDEX...................................................................................................... 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.

Dennison С. A Guide to Protein Isolation

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