| Assay Method Information | |
| | Biochemical Assay |
| Description: | The ingredients required for this purpose are mixed in a black 384-well microtiter plate with transparent base (from Greiner, catalog number 781092). The requirements in this connection for each well of the 384-well microtiter plate are 5 nM GSK3β (from Upstate, catalog number xy), 40 μM GSK3β substrate GSM (sequence H-RRRPASVPPSPSLSRHS-(pS)-HQRR, from Upstate, catalog number 2-533), 30 μM nicotinamide adenine dinucleotide NADH (Roche Diagnostics, catalog number 10107735), 50 μM adenosine triphosphate ATP (from Sigma, catalog number A7966) and 2 mM phosphoenolpyruvate (from Roche, catalog number 128112). The required reaction buffer in which the biochemical reaction takes place consists of 50 mM Trizma hydrochloride Tris-HCl pH: 7.5 (from Sigma, catalog number T3253), 5 mM magnesium chloride MgCl2 (from Sigma, catalog number M8266), 0.2 mM DL-dithiothreitol DTT (from Sigma, catalog number D9779), 2 mM ethylenediaminetetraacetic acid EDTA (from Sigma, catalog number E6758), 0.01% Triton X-100 (from Sigma, catalog number T8787) and 0.05% bovine serum albumin BSA (from Sigma, catalog number B4287). Active substances are dissolved in dimethyl sulfoxide DMSO (from Sigma, catalog number D8418) in a concentration of 10 mM. Active substances are added in serial concentrations of 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM, 0.00001 μM, 0.000001 μM to the mixtures of the biochemical reaction. As control, dimethyl sulfoxide is added instead of substance in a final concentration of 0.1%. The reaction is incubated at 30° C. for 2 hours and then the resulting fluorescence is measured in a Tecan Safire-XFLUOR4 instrument, version V4.50 (serial number 12901300283) with the specifications: measurement mode−fluorescence measured from below, extinction wavelength 340 nm, emission wavelength 465 nm, slit width extinction 5 nm, slit width emission 5 nm, gain mode 120, delay 0 μs, number of light flashes per measurement 3, and an integration time of 40 μs. |
| Affinity data for this assay | |
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