| Assay Method Information | |
| | Kinase Assay |
| Description: | CDC25C (2 ug/well) with PLKl (1 ug/well) in 20 mM Tris/HCl buffer pH 7.5, supplemented with 25 mM beta-glycerophosphate, 5 mM EGTA, 1 mM DTT and 1 mM NaVO3. Serial dilutions of test compound in assay buffer were added. Reaction was initiated by the addition of 100 uM ATP and 0.5 uCi of [gamma-32P]-ATP. The reaction mixture was incubated at 30 0C for 1 h, then stopped with 75 mM aq orthophosphoric acid, transferred onto a 96-well P81 filter plate (Whatman), dried, and the extent of CDC25C phosphorylation was assessed by scintillation counting using a Packard TopCount plate reader. The raw assay data was analysed by non-linear regression analysis parameters and IC50 values were determined using the equation: y = A + ((B- A)/(l+((C/x)+D))), where y is % inhibition, A is minimum inhibition, B is maximum inhibition, C is EC5O, and D is the slope factor. |
| Affinity data for this assay | |
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