Glutathione (GSH/GSSG/Total) Assay Kit, BioAssay(TM)

Glutathione is the major intracellular low molecular weight thiol that plays a critical role in cellular defense against oxidative stress in tissues and cells. Commercially available glutathione detection kits, such as the DTNB-enzyme cycling glutathione assay kit or the Monochlorobimane based assay kit hardly distinguish between reduced glutathione (GSH; FW: 307) and oxidized glutathione (GSSG; FW: 612). This Glutathione Detection Kit provides a unique, convenient tool for detecting GSH, GSSG, and total glutathione individually. In the assay, OPA 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 to convert GSSG to GSH. Thus, GSSG can be specifically quantified. The kit provides a unique procedure and buffer formula to eliminate protein thiol interference and to stabilize GSH and GSSG in solution. The assay is easy to perform and detects 2-400 ng/ul of GSH, GSSG or total glutathione.\n\nKit Components:\nG8123-40A: Glutathione Assay Buffer, 1x30ml\nG8123-40B: PCA (Perchloric Acid, 6N), 1x2ml\nG8123-40C: KOH (3N), 1x2ml\n*G8123-40D: OPA Probe (o-phthalaldehyde), 1x200ul\n*G8123-40E: Reducing Agent Mix, x1vial\n*G8123-40F: GSH Quencher 1x20ul\n*G8123-40G: GSH Standard (FW: 307), 1x1mg\n\nStorage and Stability:\nStore components at -20 degrees C. Kit is stable for 6 months after receipt. Reconstituted *G8123-40D, *G8123-40E, *G8123-40F, *G8123-40G are stable for 2 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. \n\nSample Collection and Storage:\nTissue and Cell Samples:\nGSH is labile and cell preparations will oxidize it rapidly. Keep all samples and reagents ice cold and work as rapidly as possible. Prepare centrifuge tubes with 20ul PCA on ice to receive samples. Homogenize 2-4 x10e6 cells or 40mg tissue on ice with 100ul of ice cold Glutathione Assay Buffer. Take 60ul of each homogenate to a prechilled tube containing PCA and vortex several seconds to achieve a uniform emulsion. Keep on ice for 5 min. Spin 2 min at 13,000xg at 4 degrees C, collect supernatant (containing glutathione) and discard the protein pellet. The sample can then be stored at

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