| Assay Method Information | |
| | ATR Kinase Activity Assay |
| Description: | The amount of phosphorylated P53 protein was determined using a time-resolved fluorescence system. Before the starting of the experiment, the following working solutions were prepared as needed: 1× reaction buffer (20 mM HEPES PH8.0, 1% glycerol, 0.01% Brij-35), dilution buffer (20 mM HEPES PH8.0, 1% glycerol, 0.01% Brij-35, 5 mM DTT and 1% BSA), stop buffer (20 mM HEPES PH8.0, 1% glycerol, 0.01% Brij-35, 250 mM EDTA), detection buffer (50 mM HEPES PH7.0, 150 mM NaCl, 267 mM KF, 0.1% sodium cholate, 0.01% Tween-20, 0.0125% sodium azide). The clinically investigational drug AZD6738 (purchased from Selleck) was used as a positive control.The specific steps of the assay are as follows:4× gradient dilution compound solutions were prepared with the 1× reaction buffer to obtain 9 different compound concentrations, and 2.5 μL of the 4× gradient dilution compound solutions are added to a 384-well assay plate (784075, Greiner). 4× p53 substrate working solution (40 nM) was prepared with the 1× reaction buffer and 2.5 μL of the 4× p53 substrate working solution was added to the 384-well assay plate. 4×ATR/ATRIP working solution (12.8 ng/μL) was prepared with the dilution buffer and 2.5 μL of the 4×ATR/ATRIP working solution was added to the 384-well assay plate. 4×ATP working solution (2 mM) was prepared with deionized water, and 2.5 μL of the 4×ATP working solution was added to the 384-well assay plate. The plate was incubated at room temperature for 30 minutes in the dark. 5 μL of the stop solution was added to the 384 assay plate. Finally, 5 μL of the detection mixture (0.09 ng/μL of Anti-phospho-p53(ser15)-K and 6 ng/μL of Anti-GST-d2) to the 384-well assay plate. After incubation at room temperature overnight, the fluorescence signals were detected using an M5e (Molecular Device) instrument (excitation wavelength of 320 nm, emission wavelengths of 620 nm and 665 nm). |
| Affinity data for this assay | |
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