Bio-Rad Bio-Plex Pro™ TGF-β Assays - Manual (Page 23)

21
Add Coupled Beads, Standards, and Samples
1. Cover unused wells with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat bottom plate.
Prewet the wells using 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) coupled beads for 30 sec at medium speed.
Pour the diluted coupled beads 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 using
the wash method of choice.
5. Gently vortex the diluted standards, blanks, samples, and controls
(if applicable) for 5 sec. Transfer 50 µl to each well of the assay plate,
changing the pipet tip after every volume transfer.
6. Cover plate with a new sheet of sealing tape and protect from light
with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 2 hr at
room temperature (RT).
Note: 850 rpm provides equivalent performance to shaker settings
recommended in previous manuals (1,100 rpm for 30 sec, 300 rpm for
incubation).
Prepare and Add Detection Antibodies
Instructions are provided for diluting the detection antibodies to a
1x concentration.
1. While the samples are incubating, use Tables 10–11 or the
Calculation Worksheet on pages 37–38 to calculate the volume
of detection antibodies and detection antibody diluent needed.
Detection antibodies should be prepared 10 min before use.