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28 Charlton and Zachariou 5. Equilibration buffer: 0.02 M K 2 HPO 4 /KH 2 PO 4 + 0.5 M NaCl pH 7.4. 6. Elution buffer: 0.05 M imidazole + 0.5 M NaCl pH 7. 7. Regeneration buffer: 0.2 M EDTA + 0.5 M NaCl pH 8. 2.2. Purification of Proteins Using IMAC Based on Non-Histidine Selection and High Ionic Strength 1. Stationary support: Chelating Sepharose FF (Amersham-Pharmacia Biotech). 2. Charge solution: 0.05 M metal salts. 3. Metal rinsing solution: 0.05 M acetic acid + 0.1 M KNO 3 . 4. Pre-equilibration buffer: none. 5. Equilibration buffer: 0.03 M morpholinoethane sulphonic acid (MES) + 0.03 M imidazole + 0.5 M NaCl pH 5.5/pH 6. 6. Elution buffer: 0.03 M MES + 0.03 M imidazole + 0.1 M K 2 HPO 4 + 0.14 M NaCl pH 5.5/pH 6. 7. Regeneration buffer: 0.2 M EDTA + 0.5 M NaCl pH 8. 8. Storage solution: 0.01 M NaOH. 2.3. Purification of Proteins Using IMAC in Pseudo-Cation Exchange Mode 1. Stationary support: Chelating Sepharose FF (Amersham-Pharmacia Biotech, UK). 2. Charge solution: 0.05 M metal salts. 3. Metal rinsing solution: 0.05 M acetic acid + 0.1 M KNO 3 . 4. Pre-equilibration buffer: none. 5. Equilibration buffer: 0.03 M MES + 0.03 M imidazole + 0.05 M NaCl pH 5.5/pH 6. 6. Elution buffer: 0.03 M HEPES + 0.03 M imidazole + 0.5 M NaCl pH 8. 7. Regeneration buffer: 0.2 M EDTA + 0.5 M NaCl pH 8. 8. Storage solution: 0.01 M NaOH. 3. Method 3.1. Purification of Proteins Using IMAC Based on Histidine Selection 1. Wash packed Cu-IDA column with 2 column volumes (CV) of metal rinsing solution, 0.2 M acetic acid pH 4 (see Note 1). 2. Wash column with 5 CV of Milli Q water. 3. Pre-wash packed Cu-IDA column with 10 CV of 0.2 M K 2 HPO 4 /KH 2 PO 4 + 0.5 M NaCl, pH 7.4. 4. Equilibrate the column with 10 CV of 20 mM K 2 HPO 4 /KH 2 PO 4 + 0.5 M NaCl, pH 7.4. 5. Confirm equilibration by measuring pH and conductivity. Continue equilibration until pH and conductivity of effluent matches equilibration buffer. 6. Load sample containing target molecule ensuring the sample pH is between pH 7 and 7.2. As a general rule, loading linear velocities should be between 10 and
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Affinity Chromatography second edit
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METHODS IN MOLECULAR BIOLOGY TM Aff
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To Tina, Emmanuella, Natalie, and A
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viii Preface Affinity tags for puri
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x Contents 10. Immobilized Metal Io
Page 22: xii Contributors Tzong-Hsien Lee
Page 26: 2 Roque and Lowe cell extract conta
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Page 46: 12 Roque and Lowe A further issue o
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Page 58: 18 Roque and Lowe 39. Burton, S.J.,
Page 62: 20 Roque and Lowe 71. Bhikhabhai, R
Page 66: I Various Modes of Affinity Chromat
Page 70: 26 Charlton and Zachariou Since the
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Page 92: 38 Kumar et al. protein (5), cellul
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Page 100: 42 Kumar et al. coordination sites
Page 104: 44 Kumar et al. 5. Repeat the preci
Page 108: 46 Kumar et al. 4. Notes 1. In synt
Page 112: 48 Kumar et al. loosely bound metal
Page 116: 50 Kumar et al. 6. Ong, E., Greenwo
Page 120: 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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