The Glutathione kit is designed to quantitatively measure GSH and GSSG present in a variety of samples. Total glutathione is the sum of the oxidized form plus the GSH. A GSH standard is provided to generate a standard curve. The kit utilizes a proprietary non-fluorescent molecule, that will covalently bind to the free thiol group on GSH to yield a highly fluorescent product. After mixing the sample or standard with and incubating at room temperature for 15 minutes, the GSH-generated signal is read at 510 nm in a fluorescent plate reader with excitation at 390 nm. Addition of a reaction mixture that converts all the GSSG into free GSH which then reacts with the excess during a second 15 minute incubation yields the signal related to Total GSH content. Glutathione (GSH) is the highest concentration non-protein thiol in mammalian cells and is present in concentrations of 0.5 to 10 mM. GSH plays a key role in many biological processes, including the synthesis of proteins and DNA, the transport of amino acids, and the protection of cells against oxidation. Harmful hydrogen peroxide cellular levels are minimized by the enzyme glutathione peroxidase (GP) using GSH as a reductant. The oxidized GSH dimer, GSSG, is formed from GSH and peroxide by the GP reaction. An important role of GSSG in the NFkappaB activating signal cascade is suggested by the fact that the potent NFkappaB inducer TPA increases intracellular GSSG levels and GSSG/GSH ratios. Blood, serum, plasma, urine, erythrocytes and cell lysate samples have been validated with this assay.
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