Bio-Rad Apoptosis Assays - Manual (Page 3)

Bio-Plex Pro Assay Quick Guide
3. For sample analysis, bring samples to a final protein concentration of
500 µg/ml by diluting with lysis dilution buffer (LDB). Further dilution may
be necessary for targets with high expression levels.
4. Centrifuge samples at 500 x g for 5 min to remove particulates prior to use.
Note: Controls are ready to use after reconstitution. No further dilution
is needed.
D. Dispensing of Reagents
1. Add 10 µl of blocker to all wells of the plate.
2. Add 30 µl of the standard, control, sample, or blank to the appropriate
well of the plate.
3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of
the beads to all wells of the plate.
4. Cover plate with plate seal and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.
5. Wash the plate three times with 100 µl 1x assay buffer.
6. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
7. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do not
aspirate after incubation.
8. Prepare the required dilution of streptavidin-PE (SA-PE) as outlined in the
following table.
Note: Volumes in the table are for an entire 96-well plate. Smaller volumes
can be prepared, provided that the dilution ratios are maintained.
9. Add 20 µl of diluted SA-PE to the required plate wells.
Volume of Volume of
SA-PE Dilution SA-PE, µl 1x Assay Buffer, µl Total Volume, µl
1:10 225 2,025 2,250