Bio-Rad Bio-Plex Pro™ Human Chemokine Assays - Manual (Page 22)

20
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube. Vortex.
Each well of the assay requires 1.25 μl of the 20x stock adjusted to a
final volume of 25 μl in detection antibody diluent.
Preparing 1x detection antibodies from 20x stock (includes 25% excess volume)
Table 9. Premixed panel or one singleplex assay.
Table 10. Mixing singleplex assays.
5. After incubating the beads, samples, standards, blank, and controls,
slowly remove and discard the sealing tape.
6. Wash the plate three times with 100 µl wash buffer per well.
7. Vortex the diluted (1x) detection antibodies at medium speed for
5 sec. Pour into a reagent reservoir and transfer 25 µl to each well
of the assay plate using a multichannel pipet.
8. Cover plate with a new sheet of sealing tape and protect from light with
aluminum foil. Incubate on shaker at 850 ± 50 rpm for 30 min at RT.
Prepare and Add Streptavidin-PE (SA-PE)
1. While detection antibodies are incubating, use Table 11 or the
Calculation Worksheet on page 36 to calculate the volume of SA-PE
and assay buffer needed to prepare a 1x stock. SA-PE should be
prepared 10 min before use.
20x Detection Detection Antibody
# of Wells Antibodies, µl Diluent, µl Total Volume, µl
96 150 2,850 3,000
48 75 1,425 1,500
20x Detection 20x Detection Detection
Antibodies, µl Antibodies, µl Antibody
# of Wells Singleplex #1 Singleplex #2 Diluent, µl Total Volume, µl
96 150 150 2,700 3,000
48 75 75 1,500 1,500