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Assay Method Information

Assay Name:  Alphascreen Assay
Description:  The PI3K AlphaScreen assay (PerkinElmer, Waltham, Mass.) measures the activity of a panel of four phosphoinositide 3-kinases: PI3Kalpha, PI3Kbeta, PI3Kgamma, and PI3Kdelta. Each of these enzymes phosphorylates the 3'-hydroxyl group on phosphatidylinositiol (4,5)-bisphosphate (PIP2) to produce phosphatidylinositol (3,4,5)-trisphosphate (PIP3). This phosphorylation activity is measured using a GST-tagged PIP3 binding protein (Echelon Biosciences, Salt Lake City, Utah), an anti-GST-tagged Acceptor bead, and streptavidin-Donor bead. The interaction of biotinylated-PIP3 analog (IP4) and the PIP3 binding protein brings both Acceptor and Donor beads together producing, upon excitation of the Donor beads at 680 nm, a singlet oxygen species leading to the luminescent AlphaScreen signal. When PIP3 is produced via phosphorylation of PIP2 by a PI3K, PIP3 competes with biotinylated-PIP3 analog (IP4) for binding to the PIP3 binding protein.
Affinity data for this assay
 

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Last update November 1, 2007
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