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Assay Method Information

Assay Name:  CDK8/Cyclin C Binding Assay
Description:  The binding assay relies on the LanthaScreen™ Eu-Kinase Binding Assay (Invitrogen. This is a kinase assay platform based on measuring the binding and displacement of an Alexa Fluor® 647 conjugate of an ATP-competitive kinase inhibitor (Kinase Tracer 236, PV5592) at a kinase active site. Binding of the tracer to the kinase is detected by addition of a europium (Eu)-labelled anti-His antibody (Invitrogen PV 5596), which specifically labels the kinase of interest. This binding results in a high degree of fluorescence resonance energy transfer (FRET), whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET.The enzyme has been purchase from Invitrogen (PV4402) as a dimer of full-length His-tagged recombinant human proteins.Assay conditions were as indicated by the kit manufacturers with the following adaptations:Assay buffer: 50 mM HEPES, pH 7.5, 1 mM EGTA, 0.01% Brij-35, 10 mM MgCl2Assay volume: 25 μlIncubation time and temperature: 60 min at 25° C.Cdk8-Cyclin C concentration: 5 nMTracer concentration: 10 nM(Eu)-labeled anti-His antibody concentration: 1.5 nMTested compound: Serial 1:3 dilutionsFinal DMSO concentration in the assay: 1%Assays were performed in 384-well plates. The final read out was generated using an EnVision plate reader (Perkin-Elmer). The emission ratio was calculated by dividing the acceptor/tracer emission (665 nm) by the antibody/donor emission (615 nm).
Affinity data for this assay
 

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Last update November 1, 2007
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