Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 2)

Assay — Day 1
Note: Make sure all assay components are at RT before use.
1. Plan the plate layout. (Section 2 of the complete instruction manual)
2. Prime wash station or prepare handheld magnetic washer. (Section 3)
3. Reconstitute Bio-Rad cell lysates in 250 μl of diH
2
O per vial. Vortex
for 5 sec and incubate at RT for 20 min. Transfer the cell lysate to a
centrifuge tube and centrifuge at 15,000 x g for 10 min at 4°C.
Protein concentration is now 200 µg/ml. (Section 4)
4. Vortex coupled beads for 30 sec and dilute to 1x in cell signaling wash
buffer as shown below. Protect from light. (Section 4)
5. Vortex the diluted (1x) beads. Add 50 μl to each well of the assay plate.
6. Wash the plate two times with 200 μl wash buffer.
7. Gather samples, Bio-Rad cell lysates, and blank (detection antibody
diluent alone). Add 50 μl to each well. Recommended total amount of
protein to add is 1–10 μg per well.
8. Cover and incubate in the dark overnight (15–18 hrs) at RT with shaking.
Note: To resuspend the beads, shake vigorously 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.
Assay — Day 2
(Section 4 unless otherwise noted)
1. Start up/warm up the Bio-Plex system (may take up to 30 min). (Section 5)
Meanwhile, bring buffers and diluents to RT.
# of Wells 20x Beads, µl Wash Buffer, µl Total Volume, µl
96 288 5,472 5,760
Bio-Plex Pro Assay Quick Guide 3