| Assay Method Information | |
| | Syk Kinase Activity Inhibitiory Experiment |
| Description: | Measure of SYK protein kinase activity was carried out by using the Caliper mobility shift assay. The compound was dissolved with DMSO and diluted with kinase buffer (20 mM HEPES pH 7.5, 0.01% Triton X-100, 5 mM MgCl2, 1 mM MnCl2, 2 mM DTT), and 5 μl of 5 times the final concentration of the compounds (10% DMSO) were added to 384 well plate. 10 μl of 2.5 fold enzyme (with SYK) solution was added and incubated at room temperature for 10 minutes, then 10 of 2.5 fold substrate (Peptide FAM-P22) and ATP) solution was added. The mixture was incubated for 30 minutes under 28° C., and 25 μl of stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) was added to terminate the reaction. Conversion rate data was read by Caliper EZ Reader II (Caliper Life Sciences). Conversion rate was converted to inhibition rate data (% inhibition rate=(max−conversion rate)/(max−min)*100). The max refers to the conversion rate of the DMSO control, and min refers to the conversion rate of the enzyme-free control. The curve was drawn with the compound concentration and inhibition rate as the horizontal and vertical coordinates, and the curve was fitted with the XLFit excel add-in version 4.3.1 Software, and IC50 was calculated. |
| Affinity data for this assay | |
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