Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 20)

13. Gather the samples, Bio-Rad cell lysate controls, and blank. Use
detection antibody diluent as the blank. Transfer 50 µl of each sample
or blank to the appropriate wells of the plate.
14. Cover with a new sheet of sealing tape and incubate in the dark
overnight (15–18 hr) at room temperature with shaking.
Note : Fully resuspend the beads/sample mixture by vigorously shaking at
900–1,100 rpm for 30 sec. Slowly ramp up to speed to avoid splashing.
Then turn down to 300–450 rpm for the specified incubation time.
DAY 2
Prepare Instrument and Wash Method
15. Start up, warm up, and calibrate the Bio-Plex system as described in
Section 5, Read the Plate. This may take up to 30 min.
16. Meanwhile bring all buffers and diluents to room temperature.
17. Prepare the wash method as described in Section 3, Prepare the Wash.
Prepare and Add Detection Antibodies
18. Use Tables 8 and 9 or the Calculation Worksheet on pages 32–33 to
calculate the volume of detection antibodies and Bio-Plex detection
antibody diluent needed. Detection antibodies should be prepared
10 min before use.
19. Add the required volume of Bio-Plex detection antibody diluent to an
appropriately sized polypropylene tube.
20. Vortex the 20x stock of detection antibodies for 15–20 sec at
medium speed, then perform a quick spin to collect the entire volume
at the bottom of the tube.
21. Dilute detection antibodies to 1x by pipetting the required volume into
the tube containing detection antibody diluent. 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.
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