| Assay Method Information | |
| | Enzymatic Assay |
| Description: | Compounds were diluted from DMSO stocks into 1× buffer (20 mM MOPS, PH 7.4, 5 mM MgCl2, 0.5 mM MnCl2, 100 uM Sodium Orthovanadate, 0.01% Triton X-100, 1 mM DTT). A typical reaction assay contained 0.01 nanomoles MEK1 kinase, 0.01 nanomoles ATP, 10 nanograms substrate. The screening assay essentially comprised four additions. 2 ul of diluted compounds were dispensed to 384 well white assay plates. 6 ul of kinase-substrate cocktail was then added to each well. 2 ul 5×ATP was subsequently added to each well to start the reaction. A top seal was applied and the plate was incubated at 22 degree avoiding light for 60 minutes. Finally, 10 ul of the Kinase Glo plus reagent was added to each well to stop the reaction. Incubated at room temperature and avoid light for ten minutes. The top seal was removed and the plate was counted by the EnVision 2104 multi labeled plate reader (PerkinElmer) with a standard luminescent program. |
| Affinity data for this assay | |
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