| Assay Method Information | |
| | ATR Enzyme Assay |
| Description: | I. Experimental Materials and Instruments1. ATR enzyme (Eurofins Pharma Discovery Services, 14-953M)2. GST-tagged P53 protein (Eurofins Pharma Discovery Services, 14-952M)3. 384-well plate (Geriner bio-one, 784075)4. U-bottom 96-well plate (Geriner bio-one, 651201)5. Anti-phospho-P53 protein antibody labeled with europium cryptate (cisbio, 61P08KAZ)6. Anti-GST antibody linked to d2 (cisbio, 61GSTDLB)7. ATP solution (Sigma, R0441)8. DTT (Sigma, D0632-259)9. HEPES (Sigma, 15630080)10. Microplate reader (Envision 2104 Multilabel Reader)II. Experimental Steps15 nM ATR enzyme, 80 nM P53 protein, 300 nM ATP (the final concentrations were 40 nM and 150 nM, respectively), and small molecule compounds of various concentrations (the final concentrations (nM) of the ten points were 2985.0, 895.5, 298.5, 110.56, 33.17, 11.06, 4.09, 1.23, 0.41 and 0.15, respectively, and the final dimethyl sulfoxide concentration was 0.498%) were mixed and incubated at room temperature for 90 minutes. 10 μL of 2× cocktail buffer was added to the mixture of ATR, compound and substrate in the assay plate (anti-phospho-p53-Eu and anti-GST-d2 were diluted in the assay buffer). The resulting mixture was centrifuged at 1000 rpm for 30 seconds, and incubated overnight at 4° C. in the dark (a total of 20 μl in each well). The FRET signal (endpoint) was measured in the Envision instrument (HTRF 665/612 ratio was calculated at 665 nm emission and 612 nm emission). Data were processed using GraphPad software. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |