Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 19)

17
7.2 Sample Protocol Using Bio-Plex COOH Beads
Notes:
a. Beads must be completely protected from light throughout this
procedure.
b. The stock bead concentration is 1.25 x 10 7 beads/ml.
c. 1x scale = 1.25 x 10 6 beads.
d. Volume stock beads required (ml) = [(1.25Ч10 6 ) Ч (scale)] ч
[1.25Ч10 7 bead/ml]
Procedure:
1. Vortex the stock uncoupled beads at speed 7 for 30 sec, then
sonicate for 15 sec.
2. For a 1x scale coupling reaction, transfer 100 µL of monodisperse
COOH beads (1.25 x 10 6 beads) to one of the coupling reaction
tubes provided with the kit.
3. Centrifuge the beads at 14,000 x g for 4 min.
4. Carefully remove and discard the supernatant from the bead pellet.
5. Resuspend the beads in 100 µL bead wash buffer by vortex for
approximately 30 sec.
6. Centrifuge the beads at 14,000 x g for 4 min.
7. Carefully remove and discard the supernatant from the bead pellet.
8. 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.