| Assay Method Information | |
| | Inhibitory Effect of Compounds Disclosed Herein on ATR Enzyme |
| Description: | The following method was used to determine the inhibitory effect of the compounds disclosed herein on ATR enzyme. The experimental method was briefly described as follows:I. Experimental Materials and Instruments1. ATR enzyme (Eurofins Pharma Discovery Services, 14-953-M)2. GST-tag P53 protein (Eurofins Pharma Discovery Services, 14-952-M)3. 384-well plate (Thermo Scientific, 267462)4. U-shaped bottom 96-well plate (Corning, 3795)5. MAb Anti-phospho p53-Eu cryptate (Cisbio, 61P08KAE)6. MAb Anti GST-d2 (Cisbio, 61GSTDLF)7. ATP solution (Promega, V916B)8. EDTA (Thermo Scientific, AM9260G)9. HEPES (Gibco, 15630-080)10. Microplate reader (BMG, PHERAsta)II. Experimental Procedures1 nM ATR enzyme, 50 nM P53 protein, 7.435 μM ATP and small molecule compounds of different concentrations (serially 3-fold diluted from 1 μM to the 11th concentration) were mixed and incubated at room temperature for 2 h. A terminating buffer (12.5 mM HEPES, 250 mM EDTA) was added. The mixture was well mixed before 0.42 ng/well of mAb anti-phospho p53-Eu cryptate and 25 ng/well of mAb anti GST-d2 were added. The mixture was incubated overnight at room temperature, and the fluorescence signals at 620 nm and 665 nm were detected using a PHERAstar system. Data were processed using GraphPad software. |
| Affinity data for this assay | |
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