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Assay Method Information |
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| | Kinase Multiscreen 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. The reaction was stopped with the addition of 10 uL of EDTA, 150 mM. An amount of 100 uL was transferred to a MultiScreen plate to allow substrate binding to phosphocellulose filter. Plates were then washed three times with 100 uL of H2PO4, 75 mM, filtered by a MultiScreen filtration system, and dried. After the additon of 100 uL of Microscint 0 (Packard), 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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