Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

100 Coupled ligand µmoles/g conjugate 75 50 25 20 mg/ml 5 mg/ml 0 9 10 11 12 13 14 pH Fig. 68. pH dependence of coupling N-acetyl-D-galactosamine to Epoxy-activated Sepharose 6B. Carbonate/bicarbonate buffers were used in the range pH 9–11, sodium hydroxide solution in the range pH 12–14. Ligand concentrations: 5 mg/ml and 20 mg/ml. When a ligand contains more than one kind of group (thiol, amino and hydroxyl), the coupling pH will determine which of these groups is coupled preferentially. As a general rule, the order of coupling is e-amino > thiol > a-amino > hydroxyl although the exact result will depend on the detailed structure of the ligand. The time of reaction depends greatly on the pH of the coupling solution, properties of the ligand and the coupling temperature. The stability of the ligand and the carbohydrate chains of the matrix limit the maximum pH that can be used. Coupling is performed in the range pH 9–13 as shown in Figure 68 and the efficiency of coupling is pH and temperature dependent (Figure 69). 100 pH 11.0, +45 °C pH 10.5, +40 °C Coupled glycylleucine % 75 50 25 0 0 8 16 24 32 40 48 Time hours Fig. 69. Efficiency of coupling glycyl-leucine to Epoxy-activated Sepharose 6B. 118
Media characteristics Product Composition pH stability* Mean particle size Epoxy-activated Sepharose 6B Sepharose 6B reacts with Short term 2–14 90 µm 1,4 bis-(2,3 epoxypropoxy-) Long term 2–14 butane to form a stable ether linkage. *Long term refers to the pH interval over which the matrix is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures. Stability data refers to the coupled medium provided that the ligand can withstand the pH. Storage Store the freeze-dried powder dry below +8 °C. Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example, PBS, 0.05% NaN 3 , pH 7.2 or 20% ethanol in a suitable buffer. The pH stability of the media when coupled to a ligand will depend upon the stability of the ligand. 119
Page 1 and 2:
Affinity Chromatography Principles
Page 3 and 4:
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: Alternative coupling solutions: Dis
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
show all

Affinity Chromatography - Department of Molecular and Cellular ...

Create successful ePaper yourself

Delete template?

Save as template?