Coupling Antibodies to Protein A or G

1. use 2 mg of antibody per ml wet beads (use appropriate antibody/protein A or G combination).

2. mix antibodies with beads and bind at room temperature for at least 1 hr (on roller).

3. wash the beads twice with 10 volumes borate buffer, spin each time 3 min at 4000 rpm.

4. resuspend beads in 10 volumes borate buffer.

5. remove equivalent of 10 µl beads (= before sample).

6. add solid DMP (dimethylpimelimidate) to a final concentration of 20 mM [52 mg for 10 ml].

7. mix on roller for 30 min at room temperature.

8. remove equivalent of 10 µl beads (= after sample).

9. stop reaction by washing the beads twice in 0.2 M ethanolamine pH 8.0.

10. incubate on roller for 2 hr at room temperature in 0.2 M ethanolamine pH 8.0.

11. wash beads twice with PBS.

12. beads can be stored in PBS at 4°C.

13. check coupling by analysing the before and the after sample on a 10% SDS gel.