Bio-Rad Human Metabolic and Hormone Assays - Manual (Page 21)

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Considerations When Using a Vacuum Manifold
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After each incubation, place the filter plate on a calibrated vacuum
apparatus and remove the liquid by vacuum filtration
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To wash, add 100 μl wash buffer to each well and remove the liquid as
before. Ensure that all wells are exposed to the vacuum
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Thoroughly blot the bottom of the filter plate with a clean paper towel
between each vacuum step to prevent cross contamination
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Place the assay plate on the plastic plate holder/tray as needed
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Before each incubation, gently cover the plate with a new plate seal.
Avoid pressing down on the wells to prevent leaking from the bottom
Assay Protocol: Dispensing of Reagents
1. Add 10 µl blocker to all wells of the plate.
2. Add 30 µl 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
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. Do not aspirate
after incubation.
5. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
6. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do
not aspirate after incubation.
7. Prepare streptavidin-PE (SA-PE) as outlined in Table 9.
Note: Volumes in the table are for an entire 96-well plate. Smaller
volumes can be prepared, provided that dilution ratios are maintained.
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 9. SA-PE dilution.