| Assay Method Information | |
| | ATR Enzyme Activity Inhibition Assay |
| Description: | Prepare various buffer systems required for the experiment:1. Prepare ATR reaction buffer containing 25 mM HEPES (Gibco, Cat #15630-080), 5 mM DTT (Sigma, Cat #D0632-10G), 10 mM MnCl2 (Sigma, Cat #7773-01-5), 5 mM DTT (Sigma, Cat #D0632-10G), 1 mg/mL of BSA (Sigma, Cat #B2064-50G), 0.01% Brij35 (Sigma, Cat #9002-92-0), 1% Glycerol (Sigma, Cat #G5516-500 ML), and H2O. The full-length ATR enzyme (eurofins, Cat #14-953M) was diluted to 60 nM using the prepared ATR buffer.2. Prepare reaction substrate containing 80 nM p53 (eurofins, Cat #14-952M) and 300 nM ATP (Sigma, Cat #R0441).3. Prepare detection buffer: Dilute each of the anti-phospho-p53-Eu cryptate (Cisbio, Cat #61P08KA) and anti-GST-d2 (Cisbio, Cat #61GSTDLB) to 1 unit using HTRF detection buffer (Cisbio, Cat #62SDBRDF).Powdered compounds were dissolved in DMSO to from a stock solution at a concentration of 10 mM. The solution of compounds to be tested at a concentration of 1M was performed a 3-fold dilution for 10 concentrations with DMSO. Then, the diluted solution was added to 384-well plate at 10.05 μL per well (containing 0.498% DMSO). 5 μL of ATR was added to each well accordingly. Each well is incubated at 25° C. for 10 min, added with 5 μL of reaction substrate, incubated at 25° C. for 90 min and added with 10 μL of detection solution. The reaction was carried out overnight, and the data at 665/615 nm were read by an Envision 2104 Multilabel Reader. |
| Affinity data for this assay | |
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