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1 Affinity Chromatography History, Perspectives, Limitations and Prospects Ana Cecília A. Roque and Christopher R. Lowe Summary Biomolecule separation and purification has until very recently steadfastly remained one of the more empirical aspects of modern biotechnology. Affinity chromatography, one of several types of adsorption chromatography, is particularly suited for the efficient isolation of biomolecules. This technique relies on the adsorbent bed material that has biological affinity for the substance to be isolated. This review is intended to place affinity chromatography in historical perspective and describe the current status, limitations and future prospects for the technique in modern biotechnology. Key Words: Affinity; chromatography; biomimetic; ligands; synthetic; proteins; purification; design; combinatorial synthesis. 1. Introduction Traditional techniques for biomolecule separation based on precipitation with pH, ionic strength, temperature, salts, solvents or polymers, ion exchange or hydrophobic chromatography are slowly being replaced by sophisticated chromatographic protocols based on biological specificity. Affinity techniques exploit highly specific biorecognition phenomena and are ideally suited to the purification of biomolecules. In affinity chromatography, the specific adsorption properties of the bed material are realized by covalently attaching the ligand complementary to the target biomolecule onto an insoluble matrix. If a crude From: Methods in Molecular Biology, vol. 421: Affinity Chromatography: Methods and Protocols, Second Edition Edited by: M. Zachariou © Humana Press, Totowa, NJ 1
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Page 6: METHODS IN MOLECULAR BIOLOGY TM Aff
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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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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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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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