| Assay Method Information | |
| | Inhibitory Activity of Compounds on ATR |
| Description: | (1) Preparation of 1 kinase buffer and reaction termination solution1) 1 kinase buffer50 mM HEPES, pH 7.50.0015% Brij-351 M MnCl22) Reaction termination solution100 mM HEPES, pH 7.50.015% Brij-350.2% Coating Reagent #350 mM EDTA(2) Preparation of test compound1) The compound was dissolved in DMSO to yield a stock solution. Before the assay, to a 96-well plate, 30 uL of a 10 mM stock solution was taken and added with 60 uL of DMSO. The solution was sequentially diluted by the way of taking 30 uL of a solution at a higher concentration to 60 uL of DMSO to yield a mixed solution at a lower concentration and transferring the mixed solution to the next well, and so on, and 10 concentration gradients were set up.2) 100 uL 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 intermediate sample plate: 40 uL of the solution in each well of the above 96-well plate was taken and transferred to a new 384-well plate as an intermediate sample plate.(3) Preparation of test plate60 nL of the solution in each well of the intermediate sample plate was taken to a test plate.(4) Kinase reaction1) ATR kinase was dissolved in the 1 kinase buffer to yield a 2 enzyme solution at a concentration that is twice the final concentration. 10 uL of the 2 enzyme solution was taken to each well of the test plate and incubated at room temperature for 10 min.2) FAM-labeled polypeptide substrate and ATP were dissolved in the 1 kinase buffer to yield a 2 substrate solution at a concentration that is twice the final concentration. 10 L of the 2 substrate solution was taken to each well of the test plate.3) Kinase reaction:After incubation at 28 C. for a certain time, 30 uL of the reaction termination solution was added to terminate the enzymatic reaction.(5) Reading of the reaction wells(6) Calculation of inhibitory rates by means of curve fitting to the read values.% inhibitory rate=(max+-sample value)/(max+-min)*100; wherein max represents the value of the total reaction well with DMSO but no compound; min represents the value of the blank control well without enzyme and compound. |
| Affinity data for this assay | |
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