Bio-Rad Human Metabolic and Hormone Assays - Manual (Page 19)

17
Assay Protocol: Dispensing of Reagents
1. Add 10 µl blocker to all wells of the plate.
2. Add 30 µl the standard, control, or sample 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
Table 7.
Note: Volumes in the table are for an entire 96-well plate. Smaller
volumes can be prepared, provided that dilution ratios are maintained.
9. Add 20 µl diluted SA-PE to the required plate wells.
10. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min at RT.
11. Wash the plate three times with 100 µl 1x assay buffer.
12. After the final wash, resuspend the beads in 100 µl assay buffer. Cover
plate as in Step 4 and shake the plate at 850 ± 50 rpm for 30 sec.
13. Remove the plate seal and read plate at low PMT (Bio-Plex ® 200),
standard PMT (Bio-Plex 3D), or default settings (Bio-Plex ® MAGPIX ™ ).
Volume of Volume of
SA-PE Dilution SA-PE, µl 1x Assay Buffer, µl Total Volume, µl
1:10 225 2,025 2,250
Table 7. SA-PE dilution.