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Contributors Marie-Isabel Aguilar • Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia José Arnau • Unizyme Laboratories A/S, Hørsholm, Denmark Katja Benčina • BIA Separations d.o.o., Ljubljana, Slovenia Mojca Benčina • Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia Natalie A. Betz • University of Wisconsin, Madison, WI Joanne L. Casey • Cooperative Research Center for Diagnostics, Department of Biochemistry, La Trobe University, Victoria, Australia Adam Charlton • Industrial Biotechnology, CSIRO Molecular and Health Technology, Australia Andrew M. Coley • Cooperative Research Center for Diagnostics, Department of Biochemistry, La Trobe University, Victoria, Australia Laurie Engel • Proteomics R&D, Promega Corporation, Fitchburg, WI Michael Foley • Cooperative Research Center for Diagnostics, Department of Biochemistry, La Trobe University, Victoria, Australia Gareth M. Forde • Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia Igor Yu. Galaev • Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Lund, Sweden Stuart R. Gallant • Process Sciences Department, BioMarin Pharmaceutical Inc, Novato, CA Becky Godat • Proteomics R&D, Promega Corporation, Fitchburg, WI Danette Hartzell • PBI R&D, Promega Biosciences Inc., San Louis Obispo, CA Niels H. H. Heegaard • Department of Autoimmunology, Statens Serum Institut, Copenhagen S, Denmark Dieter H. Klaubert • PBI R&D, Promega Corporation., Fitchburg, WI Tonny M. Johnson • Proteomics R&D, Promega Corporation, Fitchburg, WI Vish Koppaka • BioMarin Pharmaceutical Inc, Novato, CA Ashok Kumar • Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur (IITK), India Conni Lauritzen • Unizyme Laboratories A/S, Hørsholm, Denmark xi
Page 2: Affinity Chromatography second edit
Page 6: METHODS IN MOLECULAR BIOLOGY TM Aff
Page 10: To Tina, Emmanuella, Natalie, and A
Page 14: viii Preface Affinity tags for puri
Page 18: x Contents 10. Immobilized Metal Io
Page 24: 1 Affinity Chromatography History,
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Immobilized Metal Ion Affinity Chro
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38 Kumar et al. protein (5), cellul
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40 Kumar et al. Crude protein extra
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42 Kumar et al. coordination sites
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44 Kumar et al. 5. Repeat the preci
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46 Kumar et al. 4. Notes 1. In synt
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48 Kumar et al. loosely bound metal
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50 Kumar et al. 6. Ong, E., Greenwo
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52 Kumar et al. 38. Wuenschell, G.
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54 Gallant et al. monoclonal antibo
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56 Gallant et al. 10. Flush the col
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58 Gallant et al. Fig. 2. Sodium do
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60 Gallant et al. 3. Liddell, E. (2
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62 Gallant et al. Binding and eluti
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64 Gallant et al. 5. Mixing device:
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66 Gallant et al. 10. Data interpre
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68 Gallant et al. 2. Adjustment of
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6 Purification of Proteins Using Di
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Purification of Proteins Using Disp
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7 Rationally Designed Ligands for U
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Rationally Designed Ligands for Use
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112 Casey et al. be constructed by
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114 Casey et al. (i) Panning the ra
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116 Casey et al. 3. Wash the coated
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118 Casey et al. 6) Wash four times
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120 Casey et al. 3) Mix the washed
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122 Casey et al. C Absorbance 450nm
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124 Casey et al. References 1. Smit
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126 Forde Utilization of the GST-gl
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128 Forde 3. Methods 3.1. Productio
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130 Forde 4. Wash the column with 5
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132 Forde 5. BDGE is toxic (by inha
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134 Forde 250 Elution in response u
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136 Forde References 1. Wils P, Esc
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138 Charlton and Zachariou 1. Intro
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140 Charlton and Zachariou and non-
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142 Charlton and Zachariou the maxi
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144 Charlton and Zachariou Table 2
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146 Charlton and Zachariou the Tabl
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148 Charlton and Zachariou for some
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11 Methods for the Purification of
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Purification of HQ-Tagged Proteins
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12 Amylose Affinity Chromatography
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Amylose Affinity Chromatography of
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192 Urh et al. and affinity purific
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194 Urh et al. beads with HaloTag l
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196 Urh et al. 2.3. Detection of Pr
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198 Urh et al. 2. Carefully remove
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200 Urh et al. 3.2.1.2. Phase 2 Imm
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202 Urh et al. 3.2.2. Detection of
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204 Urh et al. The following protoc
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206 Urh et al. 3. Incubate for 10 m
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208 Urh et al. by 0.5% Triton X-100
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14 Site-Specific Cleavage of Fusion
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Site-Specific Cleavage of Fusion Pr
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15 The Use of TAGZyme for the Effic
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Removal of N-Terminal His-Tags 231
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16 Affinity Processing of Cell-Cont
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Affinity Processing of Cell-Contain
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258 Benčina et al. 1. Introduction
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260 Benčina et al. 3.1.1. Static I
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262 Benčina et al. [A] abs orbance
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264 Benčina et al. Table 2 Long-Te
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266 Benčina et al. Table 3 Effect
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268 Benčina et al. 8 7 n RNase 0,0
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270 Benčina et al. Table 6 Long-Te
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272 Benčina et al. B A PepC marker
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274 Benčina et al. 3. Platonova, G
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276 Forde diseases, cancer and infe
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278 Forde 12. Sample loading buffer
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280 Forde 2. Mix 100 μl of sample,
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282 Forde 12. Safety Warning: Picog
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19 Affinity Chromatography of Phosp
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Affinity Chromatography of Phosphor
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20 Protein Separation Using Immobil
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Immobilized Phospholipid Chromatogr
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21 Analysis of Proteins in Solution
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Affinity Capillary Electrophoresis
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Index Adsorption isotherm, 75, 84 A
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Index 341 Genenase I, 170, 214, 215
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Index 343 Saccharomyces cerevisae,
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