| Assay Method Information | |
| | Homogenous Time Resolved Fluorescence (HTRF) Assay |
| Description: | The homogenous time resolved fluorescence (HTRF) assay allows detection of 3,4,5-triphosphate (PIP3) formed as a result of phosphorylation of phosphotidylinositol 4,5-biphosphate (PIP2) by PI3K isoforms such as alpha, beta, gamma or delta PI3K isoform activity for alpha, beta, gamma or delta was determined using a PI3K human HTRF Assay Kit (Millipore, Billerica, Mass.) with modifications. All incubations were carried out at room temperature. Briefly, 0.5 ul of 40x inhibitor (in 100% DMSO) or 100% DMSO were added to each well of a 384-well black plate (Greiner Bio-One, Monroe, N.C.) containing 14.5ul 1x reaction buffer/PIP2 (10 mM MgCl2, 5 mM DTT, 1.38 uM PIP2) mix with or without enzyme and incubated for 10 min. After the initial incubation, 5 ul/well of 400 uM ATP was added and incubated for an additional 30 minutes. Reaction was terminated by adding 5 ul/well stop solution (Millipore, Billerica, Mass.). |
| Affinity data for this assay | |
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