| Assay Method Information | |
| | Inhibitory Activities of Compounds on PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ Kinase: ADP-Glo Kinase Assay |
| Description: | (1) Preparation of 1× kinase buffer50 mM HEPES, pH 7.53 mM MgCl21 mM EGTA100 mM NaCl0.03% CHAPS2 mM DTT(2) Preparation of test compound1) Dissolution and dilution of compound:The compound was dissolved in DMSO to yield a stock solution. Before the assay, the stock solution of the compound was diluted with DMSO in a 384-well plate to yield a 100× solution at a concentration that is 100 times a target concentration for assay.50 μL of DMSO was added, respectively, into two blank wells in the same 384 well plate, corresponding to the total reaction control without compound and the blank control without enzyme.2) Preparation of test plate: 50 nL of the compound solution in each of the above wells was taken to a test plate.(3) Kinase reaction1) PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ kinases were dissolved, respectively, in 1× kinase buffer to yield a 2× enzyme solution at a concentration that is twice the final concentration. 2.5 L of the 2× enzyme solution was taken to each well of the test plate. The blank control without enzyme was added with 2.5 μL of the 1× kinase buffer instead of the enzyme solution. The test plate was shaken for mixing evenly.2) PIP2 substrate and ATP were dissolved in the 1× kinase buffer to yield a 2× substrate solution that is twice the final concentration. 2.5 μL of the 2× substrate solution was taken to each well of the test plate. The test plate was shaken for mixing evenly.3) Kinase reactionEach well of the test plate was covered and incubated at room temperature for 1 h.(4) Kinase detection1) The ADP-Glo reagent was equilibrated at room temperature.2) 5 μL of ADP-Glo reagent was added to each well of the test plate to terminate the reaction.3) The plate was centrifuged for a short time to mix evenly, shaken gently, and equilibrated for 120 min.4) 10 μL of kinase detection reagent was added to each well, with shaking for 1 min, after equilibrating for 30 min, fluorescence detection was performed.(5) Reading of the reaction wells(6) Calculation of inhibitory rates by means of curve fitting to the read values |
| Affinity data for this assay | |
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