| Assay Method Information | |
| | EMSA |
| Description: | For IC50 measurements, a 384-well microtiter plate contained 8-point serial dilutions of inhibitors and 16-point serial dilutions of staurosporine as a reference compound. First, 4.5 μL of 2× kinase solution was added to 0.05 μL of compound (maximum concentration 1.8 mM in 90% DMSO and 10% H2O). To allow for possible slow association, plates with type II inhibitors were incubated for 60 min at 30 °C. The assay started after the addition of 4.5 μL of 2× peptide with ATP and was run for 60 min at 30 °C, after which 16 μL of stop solution was added (100 mM HEPES pH 7.5, 5% DMSO, 0.1% coating reagent (Caliper Lifescience) 10 mM EDTA, pH 8.0, 0.015% BRIJ35). All reactions were performed in 50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween 20, 0.02% BSA, 10 mM betaglycerophosphate, 0.01 mM Na3VO4, 0.6% DMSO, and 2 μM peptide. |
| Affinity data for this assay | |
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