| Assay Method Information | |
| | PI3Kα H1047R Biochemical Assay |
| Description: | Human recombinant FL H1047R mutant of PIK3α (p110α H1047R/p85α) enzyme, containing N-terminal FLAG-TEV-Avi tag on the p110α catalytic subunit, was produced as described above and assayed at Pfizer, Inc, La Jolla facility, using ADP-Glo assay kit (Promega Corp., Madison, WI). Test compounds were plated in 11 doses of 1:3 serial dilutions (100 nL volume in 100% DMSO per well) on a 384-well plate. Enzyme assay buffer (5 μL), containing 100 mM HEPES (pH 7.5), 100 mM NaCl, 20 mM MgCl2, 10 mM dithiothreitol, 0.03% CHAPS detergent, and 6 nM PIK3a H1047R enzyme was added to each well. The plate was briefly spun at 800 rpm and preincubated for 60 minutes on a shaker plate. Substrate mix (5 μL), containing 100 μM Promega Ultra Pure ATP and 100 μM 08:0 PI(4,5)P2 (1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) (ammonium salt), Avanti Polar Lipids, Inc., Alabaster, AL) was added to each well to start the reaction, the plate was briefly spun at 800 rpm and incubated for 40 minutes at controlled room temperature. The final reactions contained 3 nM enzyme, 50 μM 08:0 PI(4,5)P2, 50 μM ATP, 50 mM NaCl, 10 mM MgCl2, 5 mM dithiothreitol, 0.015% CHAPS detergent in 50 mM HEPES (pH 7.5). The reaction was stopped with ADP-Glo reagent, briefly spun at 800 rpm and incubated for 40 minutes. ADP-Glo Kinase Detection Reagent was added, and the plate was briefly spun at 800 rpm and incubated for 30 minutes. The luminescence was measured in a PHERAstar FSX plate reader (BMG Labtech, Inc., Cary, NC). |
| Affinity data for this assay | |
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