GSH GSSG Cuvette Assay
The GSH GSSG Cuvette Assay is For Research Use Only
Size: 100 Assays
Sensitivity: 0.1 µM
Standard Range: 0.1 – 3 µM
Incubation Time: 20 minutes
Sample Type: Biological Fluids
Sample Size: 200 µl
Product Developed and Manufactured in the USA
Reduced glutathione (GSH) is a tripeptide (g-glutamylcysteinylglycine) that contains a free thiol group. GSH is a major tissue antioxidant that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid hydroperoxides to their corresponding alcohols and hydrogen peroxide to water. In the GPx catalyzed reaction, the formation of a disulfide bond between two GSH molecules gives rise to oxidized glutathione (GSSG). The enzyme glutathione reductase (GR) recycles GSSG to GSH with the simultaneous oxidation of b-nicotinamide adenine dinucleotide phosphate (b-NADPH2). When cells are exposed to increased levels of oxidative stress, GSSG will accumulate and the ratio of GSH to GSSG will decrease. Therefore, the determination of the GSH/GSSG ratio and the quantification of GSSG are useful indicators of oxidative stress in cells and tissues.
SAMPLE COLLECTION AND STORAGE
The following steps are for whole blood samples. See NOTES REGARDING SAMPLES section of package insert for additional information regarding the use of whole blood and guidelines for using other sample types.
1. Add 30 µL of Scavenger to a microcentrifuge tube.
2. Carefully add 100 µL of whole blood to the bottom of the centrifuge tube and mix gently.
3. Freeze sample at –70°C; it is stable for at least 30 days.
1. Carefully add 50 µL of whole blood to the bottom of a microcentrifuge tube.
2. Freeze the sample at –70°C; it is stable for at least 30 days.
GSH / GSSG Microplate Assay Kit
Glutathione Total Assay Kit
GST Alpha ELISA Assay
The low concentration of GSSG (high GSH/GSSG ratio) in tissues coupled with the need to prevent GSH oxidation during sample preparation, are important considerations for the accurate measurement of GSSG and GSH/GSSG ratios. Guntherberg and Rost (2) first reported the use of N-ethylmaleimide (NEM) reacting with GSH to form a stable complex, therefore removing the GSH prior to the quantification of GSSG in tissues. Unfortunately, NEM inhibits GR. To overcome this problem, Griffith (3) employed 2-vinylpyridine (2-VP) to derivatize GSH. Although 2-VP does not significantly inhibit GR, it reacts relatively slowly with GSH and is not very soluble in aqueous solutions.
Scavenging of Free Thiols: The GSH / GSSG Cuvette Assay kit (Reduced/Oxidized Glutathione) employs a pyridine derivative as a thiol-scavenging reagent thereby overcoming the shortfalls of both prior methods. At the concentration employed in the assay, this derivative reacts quickly with GSH but does not interfere with the GR activity.
Thiol Quantification: The quantitative determination of the total amount of glutathione (GSH + GSSG) employs the enzymatic method first reported by Tietze (1). Briefly, the reaction of GSH with Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid (DTNB)) gives rise to a product that can be quantified spectrophotometrically at 412 nm. This reaction is used to measure the reduction of GSSG to GSH. The rate of the reaction is proportional to the GSH and GSSG concentration.
- Add 200 µL of the standards, samples, or blank to the cuvette.
- Add 200 µL of the DTNB Solution to the cuvette.
- Add 200 µL of the Reductase Solution to the cuvette.
- Mix and incubate at room temperature for 5 minutes.
- Add 200 µL of the NADPH Solution to the cuvette.
- Record the change of absorbance at 412 nm by taking readings every minute for 10 minutes. If a kinetic spectrophotometer is not available, take a reading at zero minutes and another at 10 minutes. A 405 nm filter will also work if a 412 nm is not available.
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