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        Coupling Antibodies to Protein A or G

        互联网

        1449

        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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