We get a number of questions about our very popular assays for measuring Glutathione (K006-F, K006-H). In both of these assays, the detector used to quantitate GSH in samples will also react with any free thiol group present in the sample. For this reason, proteins or other potential interfering components must first be removed. This is accomplished by treating all samples with 5% 5-sulfo-salicylic acid dihydrate (SSA), which causes larger proteins to precipitate but leaves glutathione in solution.
After centrifuging, the supernatant can be collected and diluted with Assay Buffer to a maximum of 1% SSA before testing in the assay. In the Fluorescent kit (K006-F), reduced GSH and oxidized GSSG can be determined in the same well. In the Colorimetric kit (K006-H), treating samples and standards with 2-Vinylpyridine (2VP)—a chemical that prevents reduced glutathione (GSH) from reacting with the detector—allows for the measurement of Oxidized Glutathione (GSSG) separate from any Reduced GSH present. To determine the concentration of free GSH, subtract the concentration of Oxidized GSH from the concentration of total GSH determined separately from the same samples not treated with 2VP.