Bio-Rad Affi-Gel 15 Gel - Manual (Page 13)

Any unreacted groups that remain can be blocked by
addition of a slight excess of ethanolamine at the end of
the reaction. The resulting support will have the lowest
possible residual charge.
Summary of Coupling Conditions
Concentration of ligand
Protein 25 mg/ml of gel
Low MW ligand 15-20 µmoles/ml of gel
Optimum pH
Affi-Gel 10 gel near or below pI of ligand
Affi-Gel 15 gel near or above pI of ligand
Aqueous buffers MES, MOPS, HEPES, POPSO, acetate,
bicarbonate (avoid Tris, glycine)
Organic solvents alcohols, DMSO, dioxane, acetone, for-
mamide
Temperature 4 °C recommended
Reaction time 1 - 4 hours
pH range 3-10
Reaction volume 1.5 - 4.5 ml per ml of gel bed
Other compatible buffer reducing agents such as 10 mM DTT or
components nonionic detergents
Blocking reagent ethanolamine
Suitable ligand must have primary amino group
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Shake the vial, and make sure you have a uniform sus-
pension. Transfer the desired amount to a Buchner funnel,
or glass fritted funnel. Drain the supernatant solvent, and
wash the gel with at least five bed volumes of cold iso-
propanol.
Transfer the moist gel to a test tube or flask, and add
the ligand solution. Add at least 0.5 ml of ligand solution
per ml of gel, and agitate to make a uniform suspension.
To obtain a quantitatively substituted gel with low
molecular weight ligands, it is necessary to add a slight
excess of ligand. The gel contains approximately 15
µmoles of active ester per ml of gel. In the absence of
hydrolysis, factors like time, concentration, and tempera-
ture, are less important. The reaction can be carried out at
any convenient volume at room temperature for several
hours. When using DMSO, conduct the reaction at 20 °C,
to avoid the unfavorable viscosity 4 °C.
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LIT156B 6/17/98 10:41 AM Page 20