Glutathione Colorimetric Detection Kit

1. Prepare nitrate standard curve and blanks., 2. Add 50 µL Nitrate standards to plate., 3. Add 50 µL Nitric Oxide Assay Buffer, 1X to Zero Wells., 4. Add 10 µL prepared NADH to each Well., 5. Add 10 µL prepared Nitrate Reductase (NR) to each Well., 6. Incubate 20 minutes at RT., 7. Add 25 µL Color Reagent A to each Well., 8. Incubate 5 minutes at RT., 9. Read at 540-570 nm.

4 clear half-area polystyrene 96-Well microtiter plates, #267, Oxidized Glutathione (GSSG) Standard, 250 µM, 350 µL, #6624, Colorimetric GSH Detection Substrate, 1 mL, #6625, Glutathione Assay Buffer, 225 mL, #6626, NADPH Concentrate, 1 mL, #6627, Glutathione Reductase Concentrate, 1 mL, #6622, Kit Manual

Glutathione (L-γ-glutamyl-L-cysteinylglycine; GSH, Figure 1) is the highest concentration non-protein thiol in mammalian cells and is present in concentrations of 0.5 – 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 levels of hydrogen peroxide at the cellular level 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 (Figure 2). An important role of GSSG in the NFΚß activating signal cascade is suggested by the fact that the potent NFΚß inducer, tetradecanoyl phorbol acetate, increases intracellular GSSG levels and GSSG/GSH ratios. Glutathione S-transferases (GST) are an important group of enzymes that catalyze the nucleophilic addition of GSH to electrophiles. They are encoded by 5 gene families; 4 encode cytosolic GST and 1 encodes the microsomal form of GST. They have been implicated in a number of diseases. In asthma, arachidonic acid is converted to unstable leukotriene A4 (LTA4). LTA4 is either hydrated to form LTB4 or it is conjugated to GSH by a GST, leukotriene C4 synthase, to form leukotriene C4. LTC4 and its derivative LTD4 are important molecules in bronchial asthma. Leukotriene C4 synthase is therefore an important therapeutic target. It has also been shown that increased expression of GSTs can lead to drug resistance. Three glutathione adducts of the drug melphalan, used to treat ovarian cancer and multiple myeloma, have been isolated from reactions involving human microsomal GSTs. Our Glutathione Colorimetric Detection Kit is designed to quantitatively measure glutathione (GSH) and oxidized glutathione (GSSG) present in a variety of samples. Sample types validated include: whole blood; serum; plasma; erythrocytes; urine; cell lysates; and tissue samples. No separation or washing is required. Please read the complete kit insert before performing this assay. A GSSG standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. The Reaction Mixture containing NADPH and Glutathione Reductase converts the oxidized dimer, GSSG, into Free GSH (1 GSSG = 2 GSH). The kit utilizes a Colorimetric GSH Detection Substrate that reacts with the free thiol group on GSH to yield a highly colored product. Supplied reagents are in solution and require simple dilution for use in the assay. By using 2-Vinylpyridine (2VP, not supplied) to block any Free GSH in the sample, the concentration of Oxidized Glutathione (GSSG) can be determined. Any samples that have not been treated with 2VP will yield Total GSH levels (Free GSH and GSSG). The Free GSH concentration in the sample is calculated from the difference between the Total GSH determined and the GSH generated from GSSG for the 2VP-treated samples. The concentration of GSH can be determined either as an endpoint read of the color developed at 405 nm or by measuring the rate of color development at 405 nm. Our Glutathione Fluorescent Detection Kit (Catalog Numbers #9133 and #9134) allows the measurement of both Free and Oxidized Glutathione with higher sensitivity in the same sample in the same Well without using 2-Vinylpyridine. This kit is for research use only, not for use in diagnostic procedures.