| Assay Method Information | |
| | In Vitro ATR Kinase Inhibition Assay |
| Description: | Experimental method: The inhibitory activity of the compounds on ATR kinase in vitro was determined by Mobility Shift Assay at an ATP concentration of Km using Caliper EZ Reader as a mobility shift assaying technology based on microfluidic chip technology.Experimental materials and instruments: ATR kinase (Eurofins, Cat. No. 14-953); 5-FAM-AK-17 (Gill Biochemistry, Cat. No. 524315); 1× kinase buffer (50 mM HEPES pH 7.5, 0.0015% Brij-35, 1 M MnCl2); termination buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA); Caliper EZ Reader (Caliper Life Sciences).Experimental procedure: (1) The compounds to be tested were dissolved in DMSO and serially diluted 4× for a total of 10 concentrations; (2) 0.06 μL of the compounds at different concentrations were pipetted into a 384-well plate using an Echo pipetting system; (3) a 2× kinase solution with a concentration of 10 nM ATR kinase was prepared from the ATR kinase and the 1× kinase buffer; and a 2× substrate solution with a concentration of 6 μM 5-FAM-AK-17 and 4 μM ATP was prepared from 5-FAM-AK-17, ATP and the 1× kinase buffer; (4) 10 μL of the 2× kinase solution was added into the 384-well plate and incubated for 10 min at room temperature; 10 μL of the 2× substrate solution was then added and incubated for 4 h at 28° C.; (5) 30 μL of the termination buffer was added to terminate the reaction, and then the 384-well plate was put into the Caliper EZ Reader to read the conversion rate data. |
| Affinity data for this assay | |
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