Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 17)

6. Place the tube into a magnetic separator and allow separation to
occur for 30 to 60 sec.
7. With the tube still positioned in the magnetic separator, remove the
supernatant. Take care not to disturb the beads.
8. Remove the tube from the magnetic separator and resuspend the
washed beads in 80 µL of bead activation buffer by vortex for
approximately 30 sec.
9. Add 10 µL of 50 mg/mL S-NHS (prepared in bead activation
buffer immediately prior to its use) to the beads and mix gently by
vortex.
10. Add 10 µL of 50 mg/mL EDAC (prepared in bead activation buffer
immediately prior to its use) to the beads and mix gently by vortex.
11. Cover the coupling reaction tube with aluminum foil and agitate the
beads on a shaker (or rotator) for 20 min at room temperature.
12. Add 150 µL of PBS, pH 7.4, and vortex the activated beads at high
speed for 10 sec.
13. Place the tube into a magnetic separator and allow separation to
occur for 30 to 60 sec.
14. With the tube still positioned in the magnetic separator, remove the
supernatant. Take care not to disturb the beads.
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.
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