Coupling Antibodies to Protein A or G
- use 2 mg of antibody per ml wet beads (use appropriate antibody/protein A or G combination)
- mix antibodies with beads and bind at room temperature for at least 1 hr (on roller)
- wash the beads twice with 10 volumes borate buffer, spin each time 3 min at 4000 rpm
- resuspend beads in 10 volumes borate buffer
- remove equivalent of 10 µl beads (= before sample)
- add solid DMP (dimethylpimelimidate) to a final concentration of 20 mM [52 mg for 10 ml]
- mix on roller for 30 min at room temperature
- remove equivalent of 10 µl beads (= after sample)
- stop reaction by washing the beads twice in 0.2 M ethanolamine pH 8.0
- incubate on roller for 2 hr at room temperature in 0.2 M ethanolamine pH 8.0
- wash beads twice with Pbs
- beads can be stored in Pbs at 4 °C
- check coupling by analysing the before and the after sample on a 10 % SDS gel
Buffers:
borate buffer:
0.2 M Na-borate pH 9.0
