After binding of the antibody to the beads, perform cross-linking according to the following protocol (batch protocol):
Binding buffer: TBS (50 mM Tris, 150 mM NaCl, pH 7.5)
Wash buffer: TBS with 2 M urea, pH 7.5
Elution buffer: 0.1 M glycine with 2 M urea, pH 2.9
Cross-link solutions:
- 200 mM triethanolamine, pH 8.9
- 50 mM DMP (Dimethyl pimelimidate dihydrochloride) in 200 mM triethanolamine, pH 8.9
- 100 mM ethanolamine, pH 8.9
Wash the beads with 4 gel volumes of binding buffer - centrifuge for 20 s at 12 000 x g
Change buffer: Add 4 gel volumes of triethanolamine and centrifuge for 20 s at 12 000 x g
Cross-link : Add 4 gel volumes of DMP in triethanolamine. Fully suspend the gel by manual inversion and incubate with slow, end-over-end mixing for 60 min. Centrifuge for 20 s at 12 000 x g.
Wash: Add 4 gel volumes of triethanolamine and mix by manual inversion. Centrifuge for 20 s at 12 000 x
Block: Add 4 gel volumes of ethanolamine. Mix by manual inversion and incubate end-over -end for 15 min. Centrifuge for 20 s at 12 000 x g
Remove unbound antibody: Add 4 gel volumes of elution buffer and centrifuge for 20 s at 12 000 x g.
Wash: Add 4 gel volumes of binding buffer and centrifuge for 20 seconds at 12 000 x g. Perform this step 2 times total.