Bio-Rad Human MMP and TIMP Assays - Manual (Page 19)

8. Run Assay
Considerations
n
Bring all buffers, diluents, diluted standards, diluted coupled beads, and
samples to room temperature before use
n
Use calibrated pipets and pipet carefully, avoiding bubbles. Use a new
pipet tip for every volume transfer
n
Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and
assay variability
n
Assay incubations are carried out in the dark at 850 ± 50 rpm. Cover
the plate with sealing tape and protect from light with aluminum foil
Table 7. Summary of wash steps and incubations. After each assay step, select the
appropriate Bio-Plex Pro ™ wash station program or perform the appropriate manual wash step
as summarized below.
Considerations When Using a Vacuum Manifold
n
After each incubation, place the filter plate on a calibrated vacuum
apparatus and remove the liquid by vacuum filtration
n
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
n
Thoroughly blot the bottom of the filter plate with a clean paper towel
between each vacuum step to prevent cross contamination
n
Place the assay plate on the plastic plate holder/tray as needed
n
Before each incubation, gently cover the plate with a new sheet of sealing
tape. Avoid pressing down over the wells to prevent leaking from the bottom
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Bio-Plex Pro Handheld Magnet or
Wash Station Wash Station Vacuum Manifold
Assay Step Magnetic Program Vacuum Program Manual Wash Steps
Add beads to plate MAG x2 VAC x2 2 x 100 μl
Sample incubation
Detection Ab incubation
MAG x3 VAC x3 3 x 100 μl
SA-PE incubation