Bio-Rad Bio-Plex Pro™ TGF-β Assays - Manual (Page 24)

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2. Add the required volume of Bio-Plex detection antibody diluent to a
15 ml polypropylene tube.
3. Vortex the stock detection antibodies for 15–20 sec at medium
speed, then perform a 30 sec spin to collect the entire volume at the
bottom of the tube.
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube.
Each well of the assay requires 1.25 μl (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 10. Premixed panel or one singleplex assay.
Table 11. Mixing singleplex assays.
5. After incubating the beads, samples, standards, and blank, slowly
remove and discard the sealing tape.
6. Wash the plate three times with 100 µl wash buffer.
7. Vortex the diluted (1x) detection antibodies gently for 5 sec. Pour
into a reagent reservoir and transfer 25 μl to each well 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 1 hr at room
temperature.
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 1,350 1,500