| Assay Method Information | |
| | Biochemical Assay |
| Description: | In one assay the biochemical activity IC50 values are determined with respect to inhibition of c-Kit kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested are dissolved in DMSO to a concentration of 20 mM. These are diluted 30 uL into 120 uL of DMSO (4 mM) and 1 uL is added to an assay plate. These are then serially diluted 1:2 (50 uL to 100 uL DMSO) for a total of 8 points. Plates are prepared such that each kinase reaction is 20 uL in 1x kinase buffer (25 mM HEPES, pH 7.5, 2 mM MgCl2, 2 mM MnCl2, 0.01% Tween-20, 1 mM DTT, 0.01% BSA), 5% DMSO and 100 uM ATP. Substrate is 30 nM biotin-(E4Y)10 (Millipore). C-kit kinase (obtained from Millipore (#14-559) or is prepared as described in U.S. Patent Application Publication Number 2009/0076046, the disclosure of which is hereby incorporated by reference as it relates to this assay) is at 0.75 ng per sample. |
| Affinity data for this assay | |
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