| Assay Method Information | |
| | ATR Enzyme Assay |
| Description: | In this experiment, the phosphorylation level of substrate protein P53 (Eurofins, 14-952) was detected by HTRF technology to measure the activity of ATR/ATRIP (Eurofins, 14-953) kinase. Reaction buffer (25 mM HEPES pH 8.0, 0.01% Brij-35, 1% Glycerol, 5 mM DTT, 1 mg/mL BSA), termination buffer (12.5 mM HEPES pH 8.0, 0.005% Brij-35, 0.5% Glycerol, 250 mM EDTA) and assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 267 mM KF, 0.1% sodium cholate, 0.01% Tween 20) were prepared in advance. ATR/ATRIP was diluted with reaction buffer to a working solution of 2 ng/μL, and the substrate protein P53 was diluted with reaction buffer to a working solution of 80 nM, and 4 nM of ATP (Sigma, A2383) working solution (containing 40 mM MnCl2) was prepared with reaction buffer. The compound was 3-fold diluted with DMSO, then diluted with reaction buffer into a working solution, then added to a 384-well plate at 2.5 μL/well, and centrifuged at 1500 rpm for 40 s. Then 2.5 μL of ATR/ATRIP working solution, P53 working solution and ATP working solution were added into the 384-well plate, centrifuged at 1500 rpm for 40 s and reacted at room temperature for 30 minutes. |
| Affinity data for this assay | |
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