Coupling of peptides to Sepharose beads
2. Wash the beads with PBS or PBS/0.01% Tween X-100.
3. Apply antibody solutions.
4. Wash with PBS or PBS/0.01% Tween X-100.
5. If binding in batch, transfer to a column.
6. Elute with 100 mM glycine, pH 2.5-2.8. Collecting fractions. Immediately
neutralize with 1/10 faction volume of 1 M Tris-HCl pH 8.0 in the collection
tubes.
7. Read A280 and pool antibody containing fractions.
8. Dialyze against PBS/0.02% sodium azide.
9. The affinity column can be regenerated with extensive wash of
0.2 M glycine (pH 2.8), followed by extensive wash of a pH 7.0-8 buffer.
