Gediminas Vidugiris1, Sarah Duellman1, John Shultz1, Jolanta Vidugiriene1, Hui Wang2, Jean Osterman2, Wenhui Zhou2, Poncho Meisenheimer2 and James J. Cali1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711; 2Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401
H2O2 is a reactive oxygen species (ROS) that is measured in cells as a marker of oxidative stress. It is also measured as a marker of enzyme activities that either consume or produce H2O2. It is desirable to screen chemical compounds for their capacity to alter H2O2 levels in cultured cells or for their effects on H2O2 levels in enzyme reactions. Current fluorescent assay formats are prone to false hit rates that are too high for efficient screening applications. The ROS-Glo™ luminescent H2O2 assay detects H2O2 directly, minimizes false hit rate and provides simple formats for cell-based and enzymatic assays.
Since various ROS are interconverted to H2O2 in the cell and H2O2 is the longest lived ROS, an increase in H2O2 can reflect a general increase in the ROS level. Our method for measuring hydrogen peroxide utilizes the H2O2 Substrate, which directly reacts with H2O2 to produce a luciferin precursor. Addition of the ROS-Glo™ Detection Solution converts the precursor to luciferin and provides luciferase and other components to produce a light signal proportional to the level of H2O2.