| Assay Method Information | |
| | Recombinant CDK8 Protein-Cyclin C Complex In Vitro |
| Description: | Capability of compounds of the present invention to bind to CDK8 protein was detected using LanthaScreen (ThermoFisher) assay. We detected FRET signal proportional to the amount of CDK8-bound fluorescently-labeled ligand (Tracer 236) that competes with inhibitor for ATP binding site. We carried out the measurements in a 15 μl reaction volume using a 384-well plate (Corning, #CLS4513). Enzyme CDK8/Cyclin C (ThermoFisher, #PR7261B) was mixed with antibodies Anti-His-tag-Biotin (ThermoFisher, #PV6090), Streptavidin-Eu (ThermoFisher, #PV6025), the resulting mixture was added to plate wells (5 MKπ/well). Final concentrations of substances were as follows: CDK8/Cyclin C 5 nM, Streptavidin-Eu 3 nM, Anti-His-tag-Biotin 3 nM. Staurosporine (S4400, Sigma) was used as a reference inhibitor, and 0.1% solution of dimethyl sulphoxide (DMSO) in reaction buffer was used as a blank, the reaction buffer comprised 250 mM HEPES (pH 7.5), 50 mM MgCl2, 5 mM EGTA, and 0.05% Brij-35.The inhibitors and controls in question were added to corresponding wells (5 μl/well). The plate was incubated at room temperature for 20 minutes. After incubating, 5 μl/well of tracer solution Alexa Fluor-647 (Kinase Tracer 236, ThermoFisher, #PV5592)) was added to the wells. Final concentration of tracer was 10 nM. Reaction buffer, instead of tracer solution, was used as a negative control. The plate was incubated for 40 minutes at 25 C., TR-FRET signal was then measured according to the manufacturer's guidelines on SPARK20 plate reader (Tecan, Switzerland), and the value was converted to the amount of kinase-bound tracer. |
| Affinity data for this assay | |
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