| Assay Method Information | |
| | Biochemical Assay |
| Description: | Assay-ready plates (ARPs; Proxiplate-384 PLUS, white, PerkinElmer) with compound solution in 100% DMSO are generated on an Access Labcyte Workstation with the Labcyte Echo 55x. 150 nL of compound solution are transferred per well in 11 concentrations in duplicates with serial 1:5 dilutions. The final DMSO concentration in the assay is 1%.5 nM (final assay concentration) CDK8/cyclin C, 2 nM (final assay concentration) Biotin anti-His Tag Antibody and 2 nM (final assay concentration) LanthaScreen Eu-Streptavidin are mixed in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 0.05% BSA, 1 mM DTT) prior to use and kept at room temperature.The assay runs on a fully automated robotic system. 10 μL of CDK8/cyclin C, Biotin anti-His Tag Antibody and LanthaScreen Eu-Streptavidin mix are dispensed into columns 1-24. 5 μL of 10 nM (final assay concentration) Kinase Tracer 236 solution in assay buffer are added to columns 1-23, 5 μL of assay buffer into column 24. Plates are kept at room temperature in a darkened incubator. After 60 min incubation time the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET specs from PerkinElmer. |
| Affinity data for this assay | |
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