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| | Biochemical Assay |
| Description: | PI3KA_Act: Genes encoding for full length p110alpha and p85alpha nSH-iSH2=niSH2 (p85a aminoacids 322-600) 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 niSH2 domains was subcloned into p10 promoter. Additionally, Human Rhinovirus 3C Protease (HRV 3C) site was introduced between nSH2 and iSH2, replacing aminoacids 431-438 of p85alpha with LEVLFQGP HRV 3C recognition sequence, using standard QuickChange mutagenesis protocol (Agilent Technologies, CA). Recombinant baculovirus was generated using Bac-to-Bac protocol (Life Technologies, Carlsbad, Calif.). 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. |
| Affinity data for this assay | |
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