TY - JOUR
T1 - Development and Validation of a Single-Well Cell-Based Assay for the Detection of Endogenous Phosphoproteins
AU - Sheehan, Antony
AU - Goodrich, Wendy
AU - Banks, Peter
AU - Crouch, Michael
AU - Osmond, Ronald
PY - 2013/3/1
Y1 - 2013/3/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84874913189&partnerID=8YFLogxK
U2 - 10.1089/adt.2012.471
DO - 10.1089/adt.2012.471
M3 - Article
VL - 11
SP - 108
EP - 116
JO - ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES
JF - ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES
SN - 1540-658X
IS - 2
ER -