A High Sensitivity Assay for Kinetic Analysis of Kinases and ATPases Based on Measurement of ADP Accumulation

Version:: AACR07
File Name/Number:: AACR 2007
Year:: 2007

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DiscoveRx has developed a unique assay technology based on an enzyme-coupled detection system that measures ADP accumulation which has been widely applied for kinetic studies of proteins that utilize ATP, GTP or UTP. The kinetic assay format offers a number of benefits for kinase and ATPase research in particular, including a broad tolerance to additives, no need for a modified substrate, the ability to work with whole protein substrates, no requirement for a phospho-specific antibody, rapid signal generation, constant ATP concentration, and the ability to operate the assay on any simple fluorescence-based microplate reader. The kinetic capabilities of the assay are useful for assay development and enzyme characterization experiments, as well as more details analyzes to determine compound mode of action, IC₅₀, and K_i values. Recently, DiscoveRx has made significant improvement in the technology to detect as little as 200 nM ADP in solution, while providing a S:B ratio greater than 3-fold for detection of 1-2 µM ADP, which is a commonly desired range for many screening assays...