| Assay Method Information | |
| | Inhibitory Activities of Compounds on DNA-PK Kinase: ADP-Glo Kinase Assay |
| Description: | (1) Preparation of 1× kinase buffer40 mM Tris, pH 7.50.0055% Brij-3520 mM MgCl20.05 mM DTT(2) Preparation of test compound1) The compound was dissolved in DMSO to yield a stock solution. Before the assay, the stock solution of the compound was diluted with DMSO to yield a 100× solution at a concentration that is 100 times a target concentration for assay. If the target concentration was M, the stock solution should be diluted to yield a 1 mM solution at this step.2) 100 μL of DMSO was added, respectively, into two blank wells in the same 96 well plate, corresponding to the total reaction control without compound and the blank control without enzyme.3) Preparation of test plate: 50 nL of the compound solution in each of the above wells was taken to a 384-well plate as a test plate.(3) Kinase reaction1) DNA-PK kinase was dissolved in 1× kinase buffer to yield a 2× enzyme solution that is twice the final concentration. 2.5 μL 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) The 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 3 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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