Hi I'm investigating a small molecule inhibitor against a kinase enzyme and it has an IC50 of about 20-30µM observed in the kinaseglo assay. However when I tried to test the binding using MST there was no binding observable. Am I doing something wrong for my MST? Would appreciate any suggestions or thoughts or advice!
Here are 2 possibilities:
1. The inhibition observed in the KinaseGlo assay is artifactual. This could be due to inhibition of one or more of the enzymes in the reagent, or quenching of the luciferin luminescence.
2. The binding of the compound to the enzyme has no effect on the mobility of the enzyme in the MST measurement.
You will have to try one or more additional methods to measure inhibition and/or binding of this relatively weak inhibitor. Also bear in mind that the solubility of the compound may be a limitation to measuring its interaction with the target.
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