| Assay Method Information | |
| | Evaluation of STK19 Kinase Inhibitory Activity |
| Description: | The optimal amount of STK19 is the minimal amount that produces luminescence within the linear portion of the kinase titration curve and generates an adequate signal-to-background ratio. The optimal kinase reaction time was defined as the point at which the EC50 from the enzyme titration no longer changes. The inventors finally chose 12.5 nM STK19 and 15 min as the optimal kinase concentration and reaction time for the kinase assay.The ATP titration was conducted with STK19 kinase using the enzyme concentration previously determined, and the kinase assays were performed at ATP concentrations ranging from 0.5 μM to 200 μM. The apparent ATP Km(app) was calculated by fitting data using the Michaelis-Menten equation. The ATP concentrations of 6.36 μM Km(app) for STK19 was required to show a 50% change between the maximum and minimum phosphorylated NRAS levels. |
| Affinity data for this assay | |
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