| Assay Method Information | |
| | Biochemical Assay |
| Description: | The biochemical assays of kinase activity of full-length PI3Kα (full-length p110α/p85α) or truncated PI3Kα (p110α/iSH2 p85α) were conducted using a fluorescence polarization format similar to the procedure of Yuan J., et al., (2011) PF-04691502, a Potent and Selective Oral Inhibitor of PI3K and mTOR Kinases with Antitumor Activity, Mol Cancer Ther. 10, 2189-2199. The enzymatic reactions were conducted in 50 μL volumes in 96-well plates. The reactions contained human recombinant PI3Kα (2 nM full-length p110α/p85α or 0.5 nM p110α/iSH2 p85) and 30 μM phosphatidylinositol 4,5-bisphosphate (PIP2) (Avanti Polar Lipids, Inc., Alabaster, Ala.) and were sonicated for 1 minute prior to adding PI3Kα enzyme (PI3KA_Act or PI3KA_FL), DMSO or test compound (12-point 3-fold serial dilution, 3 μM top dose, 2% DMSO final concentration), 5 mM MgCl2, 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, and 0.05% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The reactions were initiated by the addition of ATP (41 μM, Km-level, for full-length p110α/p85 or 1 mM ATP for p110α/iSH2 p85), following a 15-min preincubation. The reactions were incubated for 30 min at room temperature, stopped with EDTA pH 8 (10 mM final concentration). In a detection plate, 15 μL of detector/probe mixture, containing 480 nM GST-Grp1PH domain protein (University of Dundee, Dundee, UK) and 12 nM carboxytetramethylrhodamine (TAMRA)-tagged fluorescent phosphatidylinositol (3,4,5)-triphosphate (PIP3) (Echelon Biosciences, Inc., Salt Lake City, Utah) in assay buffer, was mixed with 15 μL of kinase reaction mixture. The plate was shaken for 30 minutes and fluorescence polarization values were measured on an LJL Analyst HT plate reader (Molecular Devices, Sunnyvale, Calif.). |
| Affinity data for this assay | |
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