Development and Validation of a Single-Well Cell-Based Assay for the Detection of Endogenous Phosphoproteins

Antony Sheehan, Wendy Goodrich, Peter Banks, Michael Crouch, Ronald Osmond

    Research output: Contribution to journalArticle

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)108-116
    Number of pages9
    JournalASSAY AND DRUG DEVELOPMENT TECHNOLOGIES
    Volume11
    Issue number2
    DOIs
    Publication statusPublished - 2013

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