Bio-Rad Bio-Plex Pro™ Human Cancer Biomarker Assays - Manual (Page 20)

18
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
Add Coupled Beads, Samples, Standards, Blank,
and Controls
1. Cover unused wells of the assay plate with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat bottom plate.
a) Prewet the wells with 100 µl assay buffer and remove the liquid
by vacuum filtration. Dry the bottom of the filter plate thoroughly
by blotting on a clean paper towel.
3. Vortex the diluted (1x) beads for 30 sec at medium speed. Pour into
a reagent reservoir and transfer 50 µl to each well of the assay plate.
TIP: A multichannel pipet is highly recommended for ease of use
and efficiency.
4. Wash the plate two times with 100 µl Bio-Plex wash buffer
according to your wash method of choice.
5. Vortex the diluted samples, standards, blank, and controls for 5 sec.
Transfer 50 µl of each to the appropriate well of the assay plate,
changing the pipet tip after every volume transfer.
6. Cover with a new sheet of sealing tape and incubate in the dark for
1 hr at room temperature with shaking at 850 ± 50 rpm.
Note! Vigorous shaking is important for optimal assay signal and
assay precision.
Prepare and Add Detection Antibodies
1. While the samples are incubating use Tables 9 and 10 or the
Calculation Worksheet on page 35 to calculate the volume of
detection antibodies and Bio-Plex detection antibody diluent needed.
Detection antibodies should be prepared 10 min before use.