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Assay Method Information |
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| | Mobility Shift Microfluidics Assay |
| Description: | Purified GSK3beta was incubated with tested compounds in doses in the presence of 4.3 uM of ATP and 1.5uM peptide substrate (Peptide 15, Caliper, MA) for 60 minutes at room temperature in 384-well plates (Seahorse Bioscience, MA), in assay buffer that contained 100 mM HEPES (pH 7.5), 10 mM MgCl2, 2.5mM DTT, 0.004% Tween-20 and 0.003% Briji-35. Reactions were terminated by the addition of 10 mM EDTA. Substrate and product were separated electrophoretically and fluorescence intensity of the substrate and product was determined by Labchip EZ Reader II (Caliper Life Sciences, MA). The kinase activity was measured as percent conversion. The reactions were performed in duplicate for each sample. Positive control, CHIR99021 was included in each plate and used to scale the data in conjunction with in-plate DMSO controls. The results were analyzed by Genedata Assay Analyzer. The percent inhibition was plotted against the compound concentration and the IC50 value was determined. |
| Affinity data for this assay | |
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