Development and validation of a single-well cell-based assay for the detection of endogenous phosphoproteins.
We describe a cellular assay for detection of phosphorylation of endogenous proteins, whereby cells are seeded, treated, and assayed for modulation of phosphorylation in a single microplate well. The procedure is coupled to a rapid, one-wash sandwich enzyme-linked immuno-sorbent assay, enabling results to be obtained within 3-4 h from cell seeding. The assay was tested in two separate cellular systems, namely, HeLa and MCF-7 cells. When using the one-well protocol with Akt phosphorylation as a model, the response to a number of agonists was the same as the response obtained using cells treated in a separate microplate, using a conventional lysate transfer approach. The assay procedure was automated, and quantitative pharmacological data on three known inhibitors of the PI3-kinase signaling pathway was obtained within 4 h from seeding cells, with six dispense steps, and a single wash cycle. Thus, the protocol affords a reliable means of assaying for cellular signaling events in different cell types, and is amenable to automation.
TGR BioSciences, Thebarton, South Australia, Australia.
Antony J
AJ
Sheehan
Wendy
W
Goodrich
Peter
P
Banks
Michael F
MF
Crouch
Ronald I W
RI
Osmond
D001681:Biological Assay / Q000295:instrumentation*; D002469:Cell Separation / Q000295:instrumentation*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation*; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism*
D010750:Phosphoproteins