| Assay Method Information | |
| | Inhibitory Activities of Compounds on ATM |
| Description: | (1) Preparation of 1× kinase basal buffer and reaction termination solution1) 1× kinase basal buffer50 mM HEPES, pH 7.50.0015% Brij-35 (polyoxyethylene lauryl ether)100 mM Na3VO45 M NaCl1 M MgCl21 M MnCl22) Reaction termination solution100 mM HEPES, pH 7.50.015% Brij-350.2% Coating Reagent #350 mM EDTA(2) Preparation of test compound1) Dissolution and dilution of compound: a compound was dissolved into DMSO to yield a 10 mM or 5 mM stock solution. To a 96-well plate, 98 μL of DMSO and 2 μL of the 10 mM stock solution were added and mixed evenly so that the concentration of the solution was 200 μM. To another 96-well plate, 45 μL of DMSO and 5 μL of the 200 μM solution were added to yield a 20 μM working solution.2) The working solution of the compound was sequentially diluted in the 96-well plate by the way of taking 10 μL of a solution at a higher concentration to 30 μL of DMSO to yield a mixed solution at a lower concentration and transferring the mixed solution to the next well, and so on, in order to set up 10 concentration gradients.3) 100 μL of DMSO was added to the blank well and served as a blank control without compound or enzyme.4) Preparation of intermediate sample plate: 40 μL of each of the solutions with gradient concentrations prepared in the 96-well plate was taken and transferred to a new 384-well plate as an intermediate sample plate.(3) Preparation of test plate100 nL of the compound solution was taken from each well of the intermediate sample plate to a 384-well plate as a test plate.(4) Kinase reaction1) The kinase was dissolved in the 1× kinase basal buffer to yield a 2× enzyme solution.2) 10 μL of the 2× enzyme solution was taken to the 384-well test plate.3) The 384-well test plate was incubated at room temperature for 10 min.4) FAM-labeled polypeptide substrate and ATP were dissolved in the 1× kinase basal buffer to yield a 2× substrate peptide solution.5) 10 μL of the 2× substrate peptide solution was taken to each well of the 384-well test plate, respectively.6) Progress and termination of enzymatic reaction: the test plate, to which the enzyme solution and the substrate peptide solution were added, was incubated at 37° C. for a while, and then 35 μL of the reaction termination solution was added to terminate the reaction.(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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