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| | c-Src Kinase Inhibitory Activity Assay |
| Description: | The kinase reaction was initiated with the incubation of the 2.5 µL of the reaction cocktail (0.7 nM of His6-Src kinase domain in kinase buffer) with 2.5 µL of prediluted compounds (dissolved in 10% DMSO, 4X target concentration) for 10 min at room temperature. The reaction cocktail contained 200 mM HEPES (pH 7.5), 16 mM MgCl2, 8 mM EGTA, 4% DMSO, 0.04% Brij-35, and 43 mM 2-mercaptoethanol. The kinase reaction was initiated by adding 5 µL of ATP/substrate (40 µM/600 µM) cocktail and incubated for 30 min at room temperature. The reaction was stopped by adding 10 µL of the 1X ADP detection mixture to the enzyme reaction mixture, followed by incubation at room temperature for 1 h. Fluorescence intensity was measured (excitation of 580 nm and emission of 630 nm). |
| Affinity data for this assay | |
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