Bio-Rad Bio-Plex Pro™ Human Cancer Biomarker Assays - Manual (Page 3)

Running the Assay
(all Section 7 in the manual unless otherwise noted)
Note: Make sure all assay components are at RT before proceeding.
1. Prewet filter plate with 100 µl Bio-Plex assay buffer (skip for flat bottom).
2. Vortex the diluted (1x) beads. Add 50 µl to each well of the assay plate.
3. Wash the plate two times with 100 µl Bio-Plex wash buffer.
4. Vortex samples, standards, blank, and controls. Add 50 µl to each well.
5. Cover and incubate in the dark for 1 hr at RT with vigorous shaking.
Note: Shake at 850 ± 50 rpm. Ramp up to speed slowly to avoid splashing.
6. With 10 min left in the incubation, vortex detection antibodies for 5 sec
and quick-spin to collect liquid. Dilute to 1x as shown below.
7. Wash the plate three times with 100 µl wash buffer.
8. Vortex the diluted (1x) detection antibodies. Add 25 µl to each well.
9. Cover and incubate in the dark for 30 min at RT. Shake at 850 ± 50 rpm.
Meanwhile, prepare Bio-Plex Manager software protocol; enter standard
S1 values provided in the assay kit. (Section 8)
10. With 10 min left in the incubation, vortex streptavidin-PE (SA-PE) for
5 sec and quick-spin to collect liquid. Dilute to 1x as shown below and
protect from light.
11. Wash the plate three times with 100 µl wash buffer.
12. Vortex the diluted (1x) SA-PE. Add 50 µl to each well.
13. Cover and incubate in the dark for 10 min at RT. Shake at 850 ± 50 rpm.
# of Wells 20x Detection Ab, µl Detection Ab Diluent, µl Total Volume, µl
96 145 2,755 2,900
Bio-Plex Pro Assay Quick Guide 1
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000