| Assay Method Information | |
| | GSK 3beta Kinase-Test |
| Description: | Human GSK3beta (expressed and prified from SF21 cells) is obtained from the University Dundee/Scotland (Dr. James Hastie Dept. of Biochemistry) in 50 mM Tris (pH7.5); 150 mM NaCl; 0.1 mM EGTA, 270 mM Succrose, 0.1% B-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF; sequence see below). The enzyme is diluted to 3.56 μM (168 μg/ml) in enzyme dilution buffer and 6 μl aliquots are stored at −80° C.The activity of GSK3 kinase protein is measured using the Z′-LYTE assay technology from Invitrogen (# PV3324).Method:The assay is performed in 384 black plates from Corning (#3676) in a final volume of 10 μl by adding 5 μl of kinase peptide mix and 2.5 μl of compound dilution. The reaction is started by addition of 2.5 μl of the 4×ATP solution.Final concentration in assay: GSK3β 5 nM, Ser/Thr9 peptide 2 μM, ATP 7 μM (ATP Km for GSK33)Positive controls are reaction mixtures containing no test compound; negative controls (blanks) are reaction mixtures containing no ATP. As a further control, the phosphopeptide solution is added to wells without kinase and without ATP (=100% phosphorylation control). The non inhibited kinase reaction will result in a phosphorylation corresponding to 20%-30% of the phosphorylation control.The reaction is performed 1 h at room temperature. After 1 h 5 μl of the development solution is added. After a further incubation for 1 h at room temperature 5 μl of the stop reagent is added. Finally the plates are read on a Flex Station II 384 (Molecular Devices).To control for any potential inhibition of the protease present in the development solution, the phosphopeptide is incubated with the development solution in the presence of the highest concentration of the test compound (usually 100 μM). |
| Affinity data for this assay | |
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