Affinity Chromatography - Department of Molecular and Cellular ...

Alternative coupling solutions:
Distilled water or aqueous buffers with sugars and carbohydrates are preferable. Carbonate, borate or phosphate
buffers can be used.
Sodium hydroxide may be used for solutions of high pH.
Organic solvents such as dimethylformamide (up to 50%) and dioxane (up to 50%) may be used to dissolve the
ligand. The same concentration of organic solvent should be included in the coupling solution.
Coupling procedure
1. Suspend the required amount of freeze-dried powder in distilled water (1 g freeze-dried powder gives about
3.0 ml final matrix volume).
2. Wash immediately for 1 hour on a sintered glass filter (porosity G3), using approximately 200 ml distilled water
per gram freeze-dried powder, added in several aliquots.
3. Dissolve the ligand in the coupling buffer to a final concentration of 0.5–10 mg/ml (for protein ligands) or
transfer solubilized ligands into the coupling buffer using a desalting column (see page 134). Adjust the pH of
the aqueous phase.
4. Use a matrix:buffer ratio of 1:2, mix the matrix suspension with the ligand solution for 16 h at +25 to +40 °C
in a shaking water bath.
5. Block remaining excess groups with 1 M ethanolamine for at least 4 h or overnight, at +40 to +50 °C.
6. Wash away excess ligand with coupling solution followed by distilled water, 0.1 M NaHCO 3 , 0.5 M NaCl,
pH 8.0 and 0.1 M NaCl, 0.1 M acetate, pH 4.0.
If organic solvents have been used, use pH paper to measure pH since solvents may damage
pH electrodes.
Using the higher temperatures can decrease coupling times.
Do not use Tris, glycine or other nucleophilic compounds as these will couple to the
oxirane groups.
Do not use magnetic stirrers as they may disrupt the Sepharose matrix.
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