METHODS IN MOLECULAR BIOLOGYTM

Protein Purification
Protocols
Second Edition

Edited by

Paul Cutler

Genomic and Proteomic Sciences, Medicines Research Center,

GlaxoSmithKline Pharmaceuticals, Stevenage, UK

Contents
Preface ... . . . . .. . . . . . . ...... . . ...... . . . ... . . . . . .... . . ...... . . . ..... . . . ...... . . .. .... . . . ..... . . . ...... . . . . .... . . . . . ..... . . . . . ... ... v

Contributors .. . . . ...... . . . ..... . . . . .. . . . . . ..... . . . .... .. . . . ... . . . .. .... . . ...... . . . ..... . . . . . .... . . . . .... . . . . . ..... . . . . . ...... .Xi
1

2
3

General Strategies
Shawn Doonan and Paul Cutler ....... ................ ................ . ........................... ......... 1
Preparation of Extracts From Animal Tissues
1. Mark Skehel .......... . . ...... ..... ........... ................................ . ........ ................... ....... 15
Protein Extraction From Plant Tissues
Roger 1. Fido, E. N. Clare Mills, Neil M. Rigby, and Peter R. Shewry ....... ......... 21

Extraction of Recombinant Protein From Bacteria

Anne F. McGettrick and D. Margaret Worrall ....... ................ ......... .......... ......... 29

Protein Extraction From Fungi

Paul D. Bridge, Tetsuo Kokubun, and Monique S.1. Simmonds ......................... 37

Subcellular Fractionation of Animal Tissues

Norma M. Ryan ........ .. ........ .. ..... ...... ... ........ . ... ........ ............................................. 47

Subcellular Fractionation of Plant Tissues : Isolation

of Plastids and Mitochondria

8
9

Alyson K. Tobin and Caroline G. Bowsher .......... .................................. ............. 53

The Extraction of Enzymes From Plant Tissues Rich in Phenolic Compounds
William S. Pierpoint .............................................. ........................ ......... ............. 65
Avoidance of Proteolysis in Extracts

Robert 1. Beynon and Simon Oliver (Revised by Paul Cutler) ....................... ..... 75

10

Concentration of Extracts
Shawn Doonan .... ....... .. .... ..... ..... ...... ... . . .......... ...... ... ...................................... ..... 85

11

Making and Changing Buffers

Shawn Doonan ........ . . . ............................................................ ........ ..................... 91

12

Purification and Concentration by Ultrafiltration

Paul Schratter (Revised by Paul Cutler) ............................................................ 101

13

Bulk Purification by Fractional Precipitation

Shawn Doonan ....... . ... ........ . .. ....... . . . . .................... . .............. .... .... .. .................... 117

14

Ion-Exchange Chromatography
Chris Selkirk ....................................................................................... .... ... ........ 125

15

Hydrophobic Interaction Chromatography

Paul A. O'Farrell .......................................................................................... ..... 133

16

Affinity Chromatography
Paul Cutler ........................................................ ............................................. ... 139

17

Dye-Ligand Affinity Chromatography

Anne F. McGettrick and D. Margaret Worrall ................................................. 151

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Contents

18 Lectin Affinity Chromatography

Iris West and Owen Goldring (Revised by Paul Cutler) . ............ .... ................. . 159

19 Immunoaffinity Chromatography

Paul Cutler ................. .. ............ . . .............. ........... ........... .. ................. ... .. . ........ . . 167

20 Immobilized Metal-Ion Affinity Chromatography

21

Tai-Tung Yip and T. William Hutchens (Revised by Paul Cutler) ... ................. . 179

Chromatography on Hydroxyapatite

Shawn Doonan . .... ... .. . .. . . . .... .... .... . .. ................. . .. .. ... ... ... . .. . . . . .. . . . .......... .............. 191

22 Thiophilic Affinity Chromatography and Related Methods

Paul Matejtschuk ............. . .. ........... ... .. .................... ............. .. .............. .. ............ 195

23 Affinity Precipitation Methods

lane A. Irwin and Keith F. Tipton ... .. ........... .......... ............ . .. .............. .. ............ 205

24 Isoelectric Focusing

Reiner Westermeier .......... .. .. ............ .. ..................... .......... .. . .............. .............. 225

25 Chromatofocusing

Timothy l. Mantle and Patricia Noone .......... ........... .............. .......................... 233

26 Size-Exclusion Chromatography

Paul Cutler . . ..................... . ...................................................................... .. ........ 239

27 Fast Protein Liquid Chromatography

David Sheehan and Siobhan O'Sullivan ................................ ........................... 253

28 Reversed-Phase Chromatography of Proteins

William A. Neville ....... ............. ................ ................... ...................................... 259

29 Extraction of Membrane Proteins

Kay Ohlendieck ......... ............... .. ............ .. ................................................... ...... 283

30 Removal of Detergent From Protein Fractions

31

Kay Ohlendieck ........... .. ..... ....... ............ .................................... .. .. .................... 295

Purification of Membrane Proteins

Kay Ohlendieck ............. .......... .. .............. ........... ............. ............. ..................... 301

32 Lyophilization of Proteins

Ciaran O'Fagain ............................. ............. . ........ . ...... . ....... .. . . . . ...... . ................. 309

33 Storage of Pure Proteins

Ciaran O'Fagain ... . ..... . . ... . . ............ ................................................. ................... 323

34 Electroelution of Proteins From Polyacrylamide Gels

Michael l. Dunn .... ........................... . ................................................ ................ 339

35 Electroblotting of Proteins From Polyacrylamide Gels

Michael l. Dunn ........................................... ..................... . . . .. . . . ..... . . ............... .. 345

36 Two-Dimensional Polyacrylamide Gel Electrophoresis

for Proteome Analyses

Neil A. lones ....................................................................... ................. ............ .. 353

37 Microscale Solution Isoelectrofocusing : A Sample Prefractionation

Method for Comprehensive Proteome Analysis

Xun Zuo and David W. Speicher ........................................................ .............. 361

38
39

Practical Column Chromatography

Shawn Doonan ........................................ . .......................... . ...... ...... ...... ........... . 377
Detection Methods

Jacek Mozdzanowski and Sudhir Burman ....... ........................... .................. . .. . 389

40

Peptide Proteomics

41

Multidimensional Liquid Chromatography of Proteins

42

Mass Spectrometry
David] . Bell ...................................... . ........ ........ ......... ....... . .......... .................. .. 447

43

Purification of Therapeutic Proteins

Julian Bonnerjea ...................................................................................... . ......... 455

44

Purification Process Scale-Up

Karl Prince and Martin Smith ....................................................... .......... .......... 463

Dean E. McNulty and]. Randall Slemmon ........................................... . ......... . . 411

Rod Watson and Tim Nadler ..... ........ . ....... ......... ........ . ....... . .......... .................. .. 425

Index ... . . . . . ..... . . . .... .. . . . . .... . . . . . ... . . . . . .... .. . . .... . . . . . ..... . . . ...... . . ...... . . . . .... . . . . . .... . . . . ...... . . . .. ... . . . . 481