Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 20)

12. Add 150 µL of PBS, pH 7.4 and vortex the activated beads at high
speed for 10 sec.
13. Centrifuge the beads at 14,000 x g for 4 min.
14. Carefully remove and discard the supernatant from the bead pellet.
15. Repeat steps 12 to 14.
16. Resuspend the activated beads with 100 µL of PBS, pH 7.4.
17. Vortex the activated beads at medium speed for 30 sec.
18. Add (5–12 µg) protein prepared in Section 6 to the activated beads
19. Bring total volume to 500 µL with PBS, pH 7.4.
20. Mix coupling reaction by vortex.
21. Incubate for 2 h at room temperature with mixing on a shaker or
rotor.
22. Centrifuge the beads at 14,000 x g for 4 min.
23. Carefully remove and discard the supernatant from the bead pellet.
24. Resuspend the coupled beads in 500 µL of PBS, pH 7.4.
25. Centrifuge the beads at 14,000 x g for 4 min.
26. Carefully remove and discard the supernatant from the bead pellet.
27. Resuspend the coupled beads with 250 µL of blocking buffer.
28. Vortex the beads at medium speed for 15 sec.
29. Cover the coupling reaction tube with aluminum foil and agitate the
beads on a shaker (or rotator) for 30 minutes at room temperature.
30. Centrifuge the beads at 14,000 x g for 4 min.
31. Carefully remove and discard the supernatant from the bead pellet.
32. Resuspend the beads in 500 µL of storage buffer by vortex 20 sec.
33. Centrifuge the beads at 14,000 x g for 6 min.
18