Affinity Chromatography - Department of Molecular and Cellular ...

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Affinity Chromatography Principles and Methods 1
Page 1: Affinity Chromatography Principles
Page 5 and 6: Serine proteases and zymogens with
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 12 and 13: Affinity chromatography is also use
Page 14 and 15: BioProcess Media for large-scale pr
Page 17 and 18: Chapter 2 Affinity chromatography i
Page 19 and 20: Avoid using magnetic stirrers as th
Page 21 and 22: pH elution A change in pH alters th
Page 23 and 24: 0.6 0.4 0.2 0 A280 pH selected for
Page 25 and 26: Situation Cause Remedy Protein does
Page 27 and 28: Chapter 3 Purification of specific
Page 29 and 30: Table 2. Relative binding strengths
Page 31 and 32: Purification examples Figure 11 sho
Page 33 and 34: MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35 and 36: Media characteristics Ligand Compos
Page 37 and 38: A 280 nm 2.0 1.5 1.0 0.5 Column: rP
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: Rec
Page 43 and 44: M r 97 000 66 000 45 000 30 000 20
Page 45 and 46: in The Recombinant Protein Handbook
Page 47 and 48: Cleaning These procedures are appli
Page 49 and 50: Purification examples Figures 24 an
Page 51 and 52: Nickel solution: 0.1 M NiSO 4 Bindi
Page 53 and 54:
Protein A fusion proteins IgG Sepha
Page 55 and 56:
Purification or removal of serine p
Page 57 and 58:
Sample: 2 ml clarified E. coli homo
Page 59 and 60:
Serine proteases and zymogens with
Page 61 and 62:
DNA binding proteins HiTrap Heparin
Page 63 and 64:
Sample: Column: Binding buffer: Elu
Page 65 and 66:
Media characteristics Ligand densit
Page 67 and 68:
Sample: Pooled and frozen human pla
Page 69 and 70:
The harsh conditions required to br
Page 71 and 72:
Purification or removal of albumin
Page 73 and 74:
IFN-act. A units 280 nm 250 0.12 20
Page 75 and 76:
Purification options Binding capaci
Page 77 and 78:
Sepharose NH (CH 2) n NH N N O N N
Page 79 and 80:
If detergent or denaturing agents h
Page 81 and 82:
Glycoproteins or polysaccharides Co
Page 83 and 84:
For complex samples containing glyc
Page 85 and 86:
For complex samples containing glyc
Page 87 and 88:
Chemical stability Avoid exposure t
Page 89 and 90:
Proteins and peptides with exposed
Page 91 and 92:
Several methods can be used to dete
Page 93 and 94:
Media characteristics Composition M
Page 95 and 96:
Performing a separation Binding buf
Page 97 and 98:
Do not store the suspension for lon
Page 99 and 100:
Sepharose has been modified and dev
Page 101 and 102:
Ligand coupling Several methods are
Page 103 and 104:
Couple a ligand through the least c
Page 105 and 106:
Table 9 summarizes recommended liga
Page 107 and 108:
Coupling through the primary amine
Page 109 and 110:
Ligand and column preparation 1. Di
Page 111 and 112:
Options Product Spacer Coupling con
Page 113 and 114:
100 80 Coupled substance, % 60 40 2
Page 115 and 116:
Coupling small ligands through amin
Page 117 and 118:
Ligand coupling 1. Add the ligand s
Page 119 and 120:
Alternative coupling solutions: Dis
Page 121 and 122:
Media characteristics Product Compo
Page 123 and 124:
Ligands containing amino groups can
Page 125 and 126:
Chapter 6 Affinity chromatography a
Page 127 and 128:
Resolution is achieved by the selec
Page 129 and 130:
For any capture step, select the te
Page 131 and 132:
Appendix 1 Sample preparation Sampl
Page 133 and 134:
Remove salts from proteins with mol
Page 135 and 136:
1. Filter (0.45 µm) or centrifuge
Page 137 and 138:
For laboratory scale operations, Ta
Page 139 and 140:
Removal of lipoproteins Lipoprotein
Page 141 and 142:
Appendix 2 Selection of purificatio
Page 143 and 144:
4. Estimate the amount of slurry (r
Page 145 and 146:
Appendix 5 Conversion data: protein
Page 147 and 148:
Middle unit residue (-H 2 0) Charge
Page 149 and 150:
Expected results when changing cond
Page 151 and 152:
Expected results with competitive e
Page 153 and 154:
Appendix 8 Analytical assays during
Page 155 and 156:
Appendix 9 Storage of biological sa
Page 157 and 158:
Ordering information Product Quanti
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
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