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3 Affinity Precipitation of Proteins Using Metal Chelates Ashok Kumar, Igor Yu. Galaev, and Bo Mattiasson Summary Metal affinity precipitation has been successfully developed as a simple purification process for the proteins that have affinity for the metal ions. The copolymers of vinylimidazole with N-isopropylacrylamide are easily synthesized by radical polymerization. When loaded with Cu(II) and Ni(II) ions, these copolymers are capable of selectively precipitating proteins with natural metal-binding groups or histidine-tagged recombinant proteins. Key Words: Metal chelate affinity precipitation; thermoresponsive copolymers; affinity macroligands; thermoprecipitation; bioseparation; recombinant histidine-tagged proteins. 1. Introduction Development of efficient and fast purification protocols in bioseparation has always been a challenging task. With the rapid advancement of gene technology, it has been possible to get any desired protein product, but the recovery of such products still poses a major problem. Affinity techniques for protein purification provide means to purify a specific protein from a complex mixture. Many affinity-based systems have been developed in recent years for the rapid purification of recombinant proteins. The methods utilize specific interactions between an affinity tag (usually a short peptide with specific molecular recognition properties, such as polyhistidines (1–3), STREP tag (4), maltose-binding From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition Edited by: M. Zachariou © Humana Press, Totowa, NJ 37
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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
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xii Contributors Tzong-Hsien Lee
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2 Roque and Lowe cell extract conta
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4 Roque and Lowe groups critical in
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6 Roque and Lowe of reinforcement e
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Page 66: I Various Modes of Affinity Chromat
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Page 92: 38 Kumar et al. protein (5), cellul
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Page 104: 44 Kumar et al. 5. Repeat the preci
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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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