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viii Preface Affinity tags for purification of proteins have become useful and common tools in academic and industrial research laboratories for rapid protein isolation. The sequencing of the human genome along with a multitude of prokaryotic genomes has forced research laboratories and biotechnology companies to find rapid and high-yielding approaches to screen for protein targets. Affinity chromatography techniques allow for high-yielding, rapid approaches to target identification. Part II presents a number of protocols describing the use of various fusion tags as well as how to cleave them, so as to allow the scientists to study the native phenotype of the protein. This section also discusses methods for selecting ligands through rational combinatorial design and phage display for use in affinity chromatography. Part III ventures into diverse applications of affinity chromatography such as its use in catalytic reactions, DNA purification, whole cell separations, and for the isolation of phosphorylated proteins. Protocols are also presented on analytical applications of affinity chromatography, such as in capillary electrophoresis and quantitative affinity chromatography. Affinity Chromatography: Methods and Protocols, Second Edition is aimed at those interested in separation sciences, particularly in the pharmaceutical and biological research sectors that have an interest in isolating macromolecules rapidly, quantitatively, and with high purity. Michael Zachariou
Page 2: Affinity Chromatography second edit
Page 6: METHODS IN MOLECULAR BIOLOGY TM Aff
Page 10: To Tina, Emmanuella, Natalie, and A
Page 16: Contents Preface ..................
Page 20: Contributors Marie-Isabel Aguilar
Page 24: 1 Affinity Chromatography History,
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Affinity Chromatography 21 85. Roqu
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2 Immobilized Metal Ion Affinity Ch
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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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