| Assay Method Information | |
| | Kinase Assay |
| Description: | The Kinase-Glo Kinase Assay Kit was purchased from Promega. All assays were performed at room temperature using white OptiPlate -384 well plate. The PI3Kα kinase was purchased from Invitrogen. The substrate was PIP2 from Invitrogen. The kinase buffer contained 50 mM Hepes (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 0.03% CHAPS and 2 mM DTT. The PI3Kα kinase solution was prepared by diluting PI3Kα kinase to 6.6 nM in kinase buffer. The substrate solution contained 100 μM PIP2 and 50 μM ATP. The test compound was diluted to 10 mM in 100% DMSO and serially diluted three times into ten different concentrations in 100% DMSO. The compound diluted in 100% DMSO was diluted 25-fold in 1×kinase buffer. 2.5 μL of the diluted compound solution and 2.5 μL PI3Kα kinase solution were added to each well of a 384-well plate. The reaction was initiated by addition of 5 μL substrate solution to each well. The final reaction volume was 10 μL, the ATP concentration was 25 μM, the PIP2 concentration was 50 μM, and the PI3Kα kinase concentration was 1.65 nM. The plate was then covered and the reaction was allowed to proceed for one hour at room temperature, followed by the addition of 10 μL Kinase-Glo reagent to each well to stop the reaction. The plate was incubated for fifteen minutes and the luminescence was read on an EnVision 2014 multilabel microplate detector plate reader. |
| Affinity data for this assay | |
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