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| | Biochemical Assay |
| Description: | PI3KA_FL: Genes encoding for full length p110alpha and p85alpha subunits of PI3Kalpha complex were subcloned from existing constructs into pFASTBAC Dual vector (Life Technologies, Carlsbad, Calif.) using standard cloning procedures. Gene encoding p110alpha subunit was subcloned into polyhedrine promoter while gene encoding p85alpha subunit was subcloned into p10 promoter. Additionally, sequence encoding for histidine tag and Tobacco Etch Virus ("TEV") cleavage site preceded p110alpha ORF (Open Reading Frame). The biochemical assays of kinase activity of full-length PI3Kalpha (full-length p110alpha/p85a) or truncated PI3Kalpha (p110alpha/iSH2 p85a) 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 uL volumes in 96-well plates. |
| Affinity data for this assay | |
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