| Assay Method Information | |
| | PI3Kα Enzymatic Assay |
| Description: | Agents: 1-fold kinase buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT). 4-fold kinase solution: PI3K ± kinase was added to 1-fold kinase buffer to form 4-fold kinase solution with a final concentration of 1.65 nM. 2-fold substrate and ATP solution: the substrate PIP2 and ATP were added to 1-fold kinase buffer to form 2-fold substrate solution with final concentration of 50 μM of PIP2 and 25 μM of ATP. 4-fold test substance solutions: 100-fold test substance solutions in different gradient concentrations were formulated with 100% DMSO, and diluted by 25 folds with 1-fold kinase buffer to form 4-fold test substance solutions in different gradient concentrations. Kinase-Glo reagent kit, which was placed to warm up to room temperature. Procedure: To each well of a 384-well plate was added 2.5 μL of the 4-fold test substance solutions in gradient concentrations. To each well was added 2.5 μL of 4-fold kinase solution, and then the plate was incubated for 10 mins. Then to each well was added 5 μL of 2-fold substrate and ATP solution, and then the plate was incubated at room temperature for 1 hour. Finally, 10 μL of the detection solution was added to terminate the reaction. After 15 mins, the data Lance signal (665 nM) was read from Envision. |
| Affinity data for this assay | |
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