Glutathione detection kit

The levels of GSH and GSH/GSSG in the brain tissue homogenates of hippocampus region were determined by using the commercially available Glutathione Assay Kit (BioVision’s Catalog #K264-100) according to the provided protocol. This BioVision’s Glutathione Detection Kit provides a unique, convenient tool for detecting GSH, GSSG, and total glutathione separately. Briefly, in the assay, OPA (o-phthalaldehyde), reacts with GSH (not GSSG), generating fluorescence, so GSH can be specifically quantified. Adding a reducing agent converts GSSG to GSH, so (GSH + GSSG) can be determined. To measure GSSG specifically, a GSH Quencher is added to remove GSH, preventing reaction with OPA (while GSSG is unaffected). Reducing agent is then added to destroy excess quencher and convert GSSG to GSH. Thus, GSSG can be specifically quantified.

Reduced glutathione (GSH), GSSG (glutathione disulfide), and total glutathione (GSH+ GSSG) in the control and null mouse hepatic tissue were determined (Glutathione Detection Kit, BioVision, Inc., Mountain View, CA)9 (link). Samples were collected in 6N perchloric acid on ice to avoid oxidation of labile GSH. GSH was determined by fluorescence of the reaction of cell samples with O-phthalaldehyde (OPA), which does not react with GSSG. GSH-GSSG total was determined by first adding a reducing agent to convert GSSG to GSH, and then reacting with OPA to produce fluorescence. To measure GSSG specifically, a GSH quencher was added initially to remove GSH, preventing the reaction with OPA, but not interacting with GSSG. Reducing agent was then added, and destroyed excess quencher and converted GSSG to GSH. Fluorescence was detected in a fluorescence plate reader (FLUOstar) with excitation and emission wavelengths of 340 nm and 420 nm, respectively.