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. |
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