Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 22)

20
27. Add the required volume of detection antibody diluent to an appropriately
sized polypropylene tube. This will be used to dilute SA-PE to 1x.
28. Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform
a quick spin to collect the entire volume at the bottom of the vial.
29. Dilute SA-PE to 1x by pipetting the required volume into the tube
containing detection antibody diluent. Vortex and protect from light
until ready to use.
Each well of the assay requires 0.5 μl of the 100x stock adjusted to a
final volume of 50 μl in detection antibody diluent.
Table 10. Preparing 1x SA-PE from 100x stock (includes 25% excess volume).
30. After detection antibody incubation, slowly remove and discard
the sealing tape.
31. Wash the plate three times with 200 µl wash buffer according to
your method of choice.
32. Vortex the diluted (1x) SA-PE at medium speed for 5 sec and
transfer 50 µl to each well of the assay plate.
33. Cover with a new sheet of sealing tape and incubate in the dark for
10 min at room temperature with shaking. Fully resuspend the beads/
SA-PE mixture by shaking at 900–1,100 rpm for 30 sec. Then turn
down to 300–450 rpm for the specified incubation time.
34. After the streptavidin-PE incubation step, slowly remove and discard
the sealing tape.
35. Wash the plate three times with 200 µl wash buffer according to
your method of choice.
# of Wells 100x SA-PE, µl Detection Antibody Diluent, µl Total Volume, µl
96 60 5,940 6,000
48 30 2,970 3,000