Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 18)

4. Run the Assay
Considerations
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Bring all assay components and samples to room temperature before use
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Use calibrated pipets and pipet carefully, avoiding bubbles
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Assay incubations are carried out in the dark. Cover the plate with
aluminum foil or otherwise protect from extended exposure to light
DAY 1
Prepare Samples and Controls
1. Thaw sample lysates and keep on ice (see Section 1, Prepare the
Samples, for lysate preparation).
2. Reconstitute lyophilized cell lysate control with 250 μl of diH
2
O,
vortex for 5 sec to mix, and incubate at room temperature for 20 min.
Protein concentration is now 200 μg/ml. Unused lysate can be stored
at –20°C for 3 months.
3. Centrifuge all samples and lysate controls at 15,000 x g for 10 min at
4°C before dispensing to wells.
Prepare and Add Coupled Beads and Samples
4. Use Tables 6 and 7 or the Calculation Worksheet on pages 32–33
as a reference to calculate the volume of coupled beads and wash
buffer needed.
5. Add the required volume of wash buffer to an appropriately-sized
polypropylene tube. This will be used to dilute beads to 1x.
6. Vortex the 20x stock of coupled beads at mid speed for 30 sec.
Carefully open the cap and pipet any liquid trapped in the cap back into
the tube. This is important to ensure maximum bead recovery. Do not
centrifuge the vial; doing so will cause the beads to pellet.
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