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

Running the Assay
Note: Make sure all assay components are at RT before pipetting.
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 plate with sealing tape and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm at RT for 1hr.
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 at 850 ± 50 rpm, as described above, in the
dark for 30 min at RT. Meanwhile, prepare Bio-Plex Manager software
protocol; enter standard S1 values and units provided in the assay kit.
10. With 10 min left in the incubation, vortex 100x 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 at 850 ± 50 rpm, as described above, in the dark
for 10 min at RT.
# of Wells 20x Detection Ab, µl Detection Ab Diluent, µl Total Volume, µl
96 150 2,850 3,000
Bio-Plex Pro Assay Quick Guide 6
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000