| Assay Method Information | |
| | TR-FRET Assay |
| Description: | The assay was performed in a white, 384 well, low volume, flat bottom plate (Grenier). After a 30 minute preincubation in assay buffer with the indicated concentration of compound, the kinase activity was initiated by the addition of the substrates. 1 nM GSK3α (GST-full length GSK3α [SignalChem G08-10G]) or 1 nM GSK3b (GST-full length GSK3b: [SignalChem G08-9G]) were then incubated at ambient temperature for 100 minutes in 10 μL of 50 mM Tris, pH 7.5, 20 mM MgCl2, 0.05 mM DTT, 100 μg/mL BSA and 1% DMSO, with 200 nM biotinylated peptide substrate (biotinylated and S645 phosphorylated glycogen synthase 631-650) and either 4 μM ATP (GSK3α, Table 16) or 2 μM ATP (GSK3b, Table 16) or 1 mM ATP (Table 17). The kinase activity was quenched with 2 μL of 250 mM EDTA. A 10 μL volume of 40 nM Strepavidin-d2, 2 nM Tb2+-pSer641 antibody in TR-FRET Detection Buffer (Invitrogen) was then added. After 60 min at room temperature the plate was read on Envision plate reader using Ex: 340 nm, Em: 615 nM and 665 nM. The normalized 665/615 Signal ratio versus log compound concentration was analyzed by XLFIT to yield IC50 values. |
| Affinity data for this assay | |
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