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Assay Method Information

Assay Name:  HTRF Kinase Inhibition Assay
Description:  The assay involves the phosphorylation of a biotinylated substrate and the detection of this phosphorylation after the addition of a streptavidin-allophycocyanin and a specific antiphosphotyrosine (or antiphosphothreonine) antibody coupled to Eu3+ cryptate. With their binding to the phosphorylated substrate the two fluorophors come into close proximity, allowing the fluorescence resonance energy transfer to occur. The proportion of substrate phosphorylated in the kinase reactions in the presence of compound compared with that phosphorylated in the presence of DMSO vehicle alone (HI control) was calculated, and the data was fitted using a Levenburg-Marquardt nonlinear regression algorithm. The inhibition constant (Ki) of the inhibitor was estimated from the IC50 using the Cheng-Prussof equation.
Affinity data for this assay
 

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Last update November 1, 2007
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