Assay Method Information

Assay Name:  Biochemical Assay for CDK7
Description:  The inhibitory activity of the test compounds was assessed by the LANCE TR-FRET assay, which detects the ATP-dependent phosphorylation of an ULight-myelin basic protein (MBP) substrate peptide (100 nM) by CDK7 (10 nM). Briefly, the enzyme reaction was run in reaction buffer (20 mM HEPES (pH 7.5), 10 mM MgCl2, 0.01% Triton x, 100 μM Sodium Orthovanadate, 1 mM DTT). The assay was performed in 384-well plate. The end concentration of the ATP substrate was 1 mM/100 μM, and that of the ULight-MBP substrate peptide was 100 nM, and of CDK7 was 10 nM. Pre-incubation of the compound and enzyme was performed for 60 min at room temperature. After 60 min incubation at room temperature, the reaction was terminated by the addition of 40 mM EDTA and 1 nM Eu-labeled anti-phospho-MBP-binding protein antibody in the buffer. Time-resolved fluorescence (excitation, 320 nm; emission donor, 615 nm; emission acceptor, 665 nm) was monitored by using 2030 multilabel reader Victor5 (PerkinElmer). The readout was calculated as (acceptor counts/donor counts)×1000. The IC50 values were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration. All fits were computed with the program Prism 5.03 (Graph Pad Software, San Diego, Calif.).
Affinity data for this assay
 

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