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| | Kinase Dowex Resin Assay |
| Description: | The biochemical activity of compounds was determined by incubation with specific enzymes and substrates, followed by quantitation of the phosphorylated product. Compounds were 3-fold serially diluted from 10 to 0.0015 uM, then incubated for 30-90 min at room temperature in the presence of ATP/P33gamma-ATP mix [2Km], substrate [5Km], and the specific enzyme [0.7-100 nM] in a final volume of 30 uL of kinase buffer, using 96 U bottom plates. An amount of 150 uL of resin/formate, pH 3.00, was added to stop the reaction and capture unreacted P33gamma-ATP, separating it from the phosphorylated substrate in solution. After 60 min of rest, a volume of 50 uL of supernatant was transferred to Optiplate 96-well plates. After the additon of 150 uL of Microscint 40 (Packard), the radioactivity was counted in the TopCount. At least two independent experiments were performed for each compound in order to determine IC50 in replicates, and potency is expressed by the mean of IC50 values obtained by nonlinear regression analysis. |
| Affinity data for this assay | |
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