GSH GSSG Microplate Assay

$515.00

The GSH GSSG Microplate Assay Kit is for the quantitative determination of the GSH/GSSG ratio in biological fluids by a microplate assay.

SKU: GSM39-K01 Categories: , ,

GSH GSSG Microplate Assay

The GSH GSSG Microplate Assay is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 0.1 µM
Standard Range: 0.1 – 3 µM
Incubation Time: 20 minutes
Sample Type: Biological Fluids
Sample Size: 500 µl
Product Developed and Manufactured in the USA


Assay Principle

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: GSH / GSSG Microplate Assay Kit 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.


Related Products

GSH GSSG Cuvette Assay
Glutathione Total Assay Kit
GST Alpha ELISA Assay

Additional Information

Assay Background


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.

Assay Procedure

  1. Add 50 µL of standards, samples, or blank to the corresponding wells on the microplate.
  2. Add 50 µL DTNB Solution to each well.
  3. Add 50 µL Reductase Solution to each well.
  4. Mix by tapping the plate or placing the plate on an orbital shaker and incubate at room temperature for 5 minutes.
  5. Add 50 µL NADPH Solution to each well.
  6. Place the plate in a kinetic plate reader and record the change of absorbance at 412 nm by taking readings every minute for 10 minutes.  If a kinetic plate reader 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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Citations


Using Cell Culture Samples:

Pettit, LK;Varsanyi, C;Tadros, J;Vassiliou, E;, Modulating the inflammatory properties of activated microglia with Docosahexaenoic acid and Aspirin, Lipids Health Dis, V. 12, Page 16, 2013

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