| Assay Method Information | |
| | ATR Inhibition Assay |
| Description: | Detection of ATR kinase activity utilized the Mobility shift assay to measure the phosphorylation of the substrate protein FAM-RAD17 (GL, Cat. No. 514318, Lot. No. P19042-MJ524315). The assay was developed and conducted at Chempartner. All the test compounds were dissolved in 100% DMSO at concentration of 20 mM, then prepare compounds and conducted the assay as follows: 1) Transfer 80 μl 20 mM compound to 40 μl of 100% DMSO in a 96-well plate.2) Serially dilute the compound by transferring 20 μl to 60 μl of 100% DMSO in the next well and so forth for a total of 10 concentrations.3) Add 100 μl of 100% DMSO to two empty wells for no compound control and no enzyme control in the same 96-well plate. Mark the plate as source plate.4) Transfer 40 μl of compound from source plate to a new 384-well plate as the intermediate plate.5) Transfer 60 nl compounds to assay plate by Echo.6) Add ATR kinase (Eurofins, Cat. No. 14-953, Lot. No. D14 JP007N) into Kinase base buffer (50 mM HEPES, pH 7.5; 0.0015% Brij-35; 0.01% Triton) to prepare 2 enzyme solution, then add 10 μl of 2 enzyme solution to each well of the 384-well assay plate, incubate at room temperature for 10 min.7) Add FAM-RAD17 and ATP (Sigma, Cat. No. A7699-1G, CAS No. 987-65-5) in the kinase base buffer to prepare 2 peptide solution, then add 10 μl to the assay plate.8) Incubate at 28 C. for specified period of time. Add 40 μl of stop buffer (100 mM HEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTA) to stop reaction.9) Collect data on Caliper. Convert conversion values to inhibition values.Percent inhibition=(max−conversion)/(max−min)*100wherein max stands for DMSO control; min stands for low control.[1373]Fit the data in XLFit excel add-in version 5.4.0.8 to ob |
| Affinity data for this assay | |
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