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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
 
Last Modified: Aug 24, 2000


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