Affinity Chromatography - Department of Molecular and Cellular ...

More documents

Recommendations

Info

Preparation of coupling reagent Use a water-soluble carbodiimide such as N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-cyclohexyl-N'-2-(4'-methyl-morpholinium) ethyl carbodiimide p-toluene sulphonate (CMC). These two carbodiimides have been used in a variety of experimental conditions and at a wide range of concentrations (Table 10). EDC often gives better coupling yields than CMC. Table 10. Examples of conditions used during coupling via carbodiimides. Coupled ligand Carbodiimide Conc. of carbodiimide pH Reaction time mg/ml Methotrexate EDC 18 6.4 1.5 h UDP-glucuronic acid EDC 32 4.8 24 h p-amino-benzamidine CMC 2 4.75 5 h Folic acid EDC 5 6 2 h Mannosylamine EDC 19 4.5–6.0 24 h Use a concentration of carbodiimide greater than the stoichiometric concentration, usually 10–100 times greater than the concentration of spacer groups. The coupling reaction is normally performed in distilled water adjusted to pH 4.5–6.0 to promote the acid-catalyzed condensation reaction. Blocking agents are not usually required after the coupling reaction if excess ligand has been used. Always use freshly prepared carbodiimides. Coupling buffer: Dissolve the carbodiimide in water and adjust to pH 4.5 Wash buffer: 0.1 M acetate, 0.5 M NaCl, pH 4 Avoid the presence of amino, phosphate or carboxyl groups, as these will compete with the coupling reaction. Preparation of EAH and ECH Sepharose 4B Wash the required amount of matrix on a sintered glass filter (porosity G3) with distilled water adjusted to pH 4.5 with HCl, followed by 0.5 M NaCl (80 ml in aliquots/ml sedimented matrix). Ligand preparation Dissolve the ligand and adjust to pH 4.5. The optimal concentration depends on the ligand. Organic solvents can be used to dissolve the ligand, if necessary. If using a mixture of organic solvent and water, adjust the pH of the water to pH 4.5 before mixing it with the organic solvent. Solvents such as dioxane (up to 50%), ethylene glycol (up to 50%), ethanol, methanol and acetone have been used. If organic solvents have been used, use pH paper to measure pH since solvents may damage pH electrodes. 114
Ligand coupling 1. Add the ligand solution followed by the carbodiimide solution to the matrix suspension and leave on an endover-end or similar mixer. Use a matrix: ligand solution ratio of 1:2 to produce a suspension that is suitable for coupling. Typically the reaction takes place overnight either at +4 °C or room temperature. 2. Adjust the pH of the reaction mixture during the first hour (pH will decrease) by adding 0.1 M sodium hydroxide. 3. Wash at pH 8 and pH 4 to remove excess reagents and reaction by-products. If a mixture of aqueous solution and organic solvent has been used, use this mixture to wash the final product as in Step 3. After Step 3 wash in distilled water, followed by the binding buffer to be used for the affinity purification. Do not use magnetic stirrers as they may disrupt the Sepharose matrix. Media characteristics Product Composition pH stability* Mean particle size EAH Sepharose 4B Covalent linkage of 1,6-diamino-hexane by Short term 3–14 90 µm epoxy coupling creates a stable, uncharged Long term 3–14 ether link between a 10-atom spacer arm and Sepharose 4B. ECH Sepharose 4B Covalent linkage of 6-aminohexanoic acid by Short term 3–14 90 µm epoxy coupling creates a stable, uncharged Long term 3–14 ether link between the 9-atom spacer arm and Sepharose 4B. *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 pre-activated matrices at +4 to +8 °C in 20% ethanol. 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. Performing a separation See page 104 for a preliminary separation protocol and Chapter 2 for general guidelines. 115
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: Coupling small ligands through amin
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
show all

Affinity Chromatography - Department of Molecular and Cellular ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?