Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 10)

8
Detailed Instructions
The following pages provide detailed instructions for each step of the assay
procedure, including sample preparation, running the assay, and reading
the plate with Bio-Plex Manager
™ and Luminex xPONENT software.
1. Prepare the Samples
Considerations
n
The degree of phosphorylation of a given analyte is highly dependent
on the cell type and cell stimulation or treatment conditions
n
Cell lines may vary in their signaling responses to the same stimulation
n
The suggested final protein concentration range in the assay is
3–200 μg/ml (0.15–10 μg per assay well) except for Pl3K p85 (Tyr 458 )
which is 31–1,000 μg/ml (1.6–50 µg/well)
n
Optimization of cell lysate concentration may be needed based on
target protein expression levels
n
Cell lysate should be clear of particulate matter before use
Cell Lysates
The Bio-Plex Pro ™ cell signaling reagent kit (catalog #171-304006M)
is required for preparing lysates derived from cell culture and tissue
samples. Just before use, prepare an adequate volume of cell lysis buffer
by adding PMSF and cell lysis factor QG.
n
Prepare 500 mM PMSF by dissolving 0.436 g PMSF in 5 ml DMSO.
(Store as aliquots at –20°C). Add PMSF to the cell lysis buffer at a final
concentration of 2 mM
n
Reconstitute cell lysis factor QG to 100x with 250 μl of diH
2
O and vortex
to mix. Add the reconstituted factor to the cell lysis buffer to a final 1x
working concentration