Affinity Chromatography - Department of Molecular and Cellular ...

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.
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