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 hour (on roller).

  3. 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.

  4. Resuspend beads in 10 volumes of 0.2 M Na-borate, pH 9.0 .

  5. Remove equivalent of 10 ul beads .

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

  7. Mix on roller for 30 minutes at room temperature.

  8. Remove equivalent of 10 ul beads.

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

  10. Incubate on roller for 2 hours at room temperature in 0.2 M ethanolamine pH 8.0.

  11. Wash beads twice with PBS buffer.

  12. Beads can be stored in PBS buffer at 4oC.

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


PBS BUFFER

TO MAKE 1L: 

  • 8 g NaCl
  • 0.2 g KCl
  • 1.15 g Na2HPO4-7H2O
  • 0.2 g KH2PO4
  • distilled H2O to 1L