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 hour (on roller).
Wash the beads twice with 10 volumes of 0.2 M Na-borate, pH 9.0; spin each time for 3 minutes at 4000 rpm.
Resuspend beads in 10 volumes of 0.2 M Na-borate, pH 9.0 .
Remove equivalent of 10 ul beads .
Add solid DMP (dimethylpimelimidate) to a final concentration of 20 mM [52 mg for 10 ml].
Mix on roller for 30 minutes at room temperature.
Remove equivalent of 10 ul beads.
Stop reaction by washing the beads twice in 0.2 M ethanolamine pH 8.0.
Incubate on roller for 2 hours at room temperature in 0.2 M ethanolamine pH 8.0.
Wash beads twice with PBS buffer.
Beads can be stored in PBS buffer at 4oC.
Check coupling by analysing the before and the after sample on a 10% SDS gel.
TO MAKE 1L:
- 8 g NaCl
- 0.2 g KCl
- 1.15 g Na2HPO4-7H2O
- 0.2 g KH2PO4
- distilled H2O to 1L