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

18
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.
Prepare and Add Detection Antibodies
1. While the samples are incubating use Table 8 or the Calculation
Worksheet on page 34 to calculate the volume of detection
antibodies and Bio-Plex detection antibody diluent needed. Detection
antibodies should be prepared 10 min before use.
2. Add the required volume of Bio-Plex detection antibody diluent to a
15 ml polypropylene tube.
3. Vortex the 20x stock of detection antibodies for 15 sec at medium
speed, then perform a 30 sec spin to collect the entire volume at the
bottom of the tube.