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ADP Hunter assay for HTS of kinase inhibitors using the PHERAstar

Lindy Kauffman. Uyen Hyugen DiscoverX Corporation 05/2006

As mediators of eukaryotic signal transduction, controlling multiple cellular processes such as gene transcription, cell cycling, migration, apoptosis and differentiation, protein kinases are considered important targets in drug discovery. Kinases phosphorylate their substrates by using ATP as a phosphate donor and generating ADP. DiscoverX has developed a homogeneuos fluorescence based assay to measure the generation of ADP. The assay uses an enzyme-coupled reaction that produces a red-shifted fluorescence signal that is directly proportional to the amount of ADP in the solution.

 

A standard curve of ADP was measured in 384 well format to validate the assay on the PHERAstar® microplate reader. Measuring ADP from 1.5 to 120 µM resulted in signal to background ratios >10 and Z' values >0.7, indicative of a robust assay-reader combination for screening compounds in high-throughput.

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