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ATR kinase assay
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ATR for use in the in vitro enzyme assay was obtained from HeLa nuclear extract by immunoprecipitation with rabbit polyclonal antiserum raised to amino acids 400 ~ 480 of ATR contained in the following buffer: 25 mM HEPES (pH 7.4), 2 mM MgCl2, 250 mM NaCl, 0.5 mM EDTA, 0.1 mM Na3VO4, 10% v/v glycerol, and 0.01% v/v Tween 20. ATR-antibody complexes were isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 hr and then through centrifugation to recover the beads. In the well of a 96-well plate, 10 μL ATR-containing Sepharose beads were incubated with 1 μg of substrate glutathione S-transferase-p53N66 in ATR assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3VO4, 0.1 mM DTT, and 10% (v/v) glycerol) at 37°C in the presence or absence of inhibitor. After 10 mins with gentle shaking, ATP was added to a final concentration of 3 μM and the reaction continued at 37°C for an additional 1 hr. The reaction was stopped by addition of 100 μL of PBS, and the reaction was transferred to a white opaque glutathione coated 96-well plate and incubated overnight at 4°C. This plate was then washed with PBS/0.05% (v/v) Tween 20, blotted dry, and analyzed by a standard ELISA technique with a phosphoserine 15 p53 antibody. The detection of phosphorylated glutathione S-transferase?p53N66 substrate was performed in combination with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence solution was used to produce a signal, and chemiluminescent detection was carried out via a TopCount plate reader. The resulting calculated % enzyme activity was then used to determine the IC50 values for the compounds.
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References:
[1]. Foote KM, Blades K, Cronin A et al. Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6- [1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity. J Med Chem. 2013 Mar 14;56(5):2125-38.
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