| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | 1× kinase base buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35) and a stop buffer (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) were prepared for testing kinases. The compounds were diluted with 100% DMSO to 100 times the highest final inhibitor concentration required in the reaction. 100 μl of dilution of the compound was transferred to wells of a 96-well plate. For example, 100000 nM solution of the compound in DMSO was prepared in this step in case that an inhibitor concentration of 1000 nM is required. 100 μl of 100% DMSO was added to two empty wells of a 96-well plate for no compound control and no enzyme control. This plate was labeled as the source plate. A new 96-well plate with 5 μl of compound transferred from the source plate is used as an intermediate plate. 95 μl of 1× kinase buffer was added to each well of the intermediate plate. The compounds in the intermediate plate was mixed on a shaker for 10 minutes. 5 μl of sample in each well from the 96-well intermediate plate was transferred to the 384-well plate in duplicate. For example, well A1 of the 96-well plate was transferred to wells A2 and A1 of the 384-well plate, and well A2 of the 96-well plate was transferred to wells A3 and A4 of the 384-well plate, and so forth. 2.5× enzyme solution was prepared: 1× kinase basal buffer with added kinase. 2.5× peptide solution was prepared: 1× kinase basal buffer with added FAM-labeled peptide and ATP. To an assay plate already contained 5 μl of 10% dimethyl sulfoxide compound, 2.5× enzyme solution was transferred, and each well of the 384-well assay plate was added 10 μl of 2.5× enzyme solution. After incubation at room temperature for 10 minutes, 2.5× peptide solution was transferred to the assay plate. Each well of the 384-well assay plate was added 10 μl of 2.5× peptide solution. Kinase reaction and termination: incubation at 28° C. for a specified period followed by the addition of 25 μl stop buffer to stop the reaction. |
| Affinity data for this assay | |
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