Cardiovascular and Oxidative Stress Assay Kit

Glutathione Total Assay Kit

$435.00

The Total Glutathione Assay kit is for the quantitative determination of Glutathione in biological fluids by a microplate assay.

SKU: GLU39-K01 Categories: , ,

Glutathione Total Assay Kit

The Glutathione Total Assay Kit is For Research Use Only

Size: 1 x 96 wells
Sensitivity: 0.5 µM
Standard Range: 0.5 – 20 µM
Incubation Time: 20 minutes
Sample Type: Whole Blood, Erythrocyte Lysate, Hepatocyte Lysate, Tissue Extract, Urine, Saliva, Cell Cultures
Sample Size: 50 µl

Assay Background

Glutathione (g-glutamylcysteinylglycine or GSH) is a naturally occurring tripeptide whose nucleophilic and reducing properties play a central role in metabolic pathways, as well as in the antioxidant system of most aerobic cells. GSH is required as a coenzyme by a variety of enzymes including glutathione peroxidase, glutathione S-transferase and thioltransferase. GSH also plays a major role in drug metabolism, calcium metabolism, the g-glutamyl cycle, cell membrane and blood platelet functions. GSH is crucial to a variety of life processes, including the detoxification of xenobiotics, maintenance of the –SH level of proteins, thiol-disulfide exchange, removal of hydroperoxides and free radicals, and amino acid transport across membranes. Physiological values for the concentration of intracellular GSH generally range from 1 to 10 mM. Although many methods have been described for the assay of GSH, most of the reliable ones have been labor intensive.

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Additional Information

Assay Principle

The Total Glutathione (tGSH) Microplate Assay kit employs a kinetic enzymatic recycling assay based on the oxidation of GSH by 5,5’-dithiobis(2-nitrobenzoic acid) [DTNB] to measure the total glutathione (tGSH) content of biological samples. The included glutathione standards or treated samples are added to the microtiter plate wells, followed by DTNB and glutathione reductase. Addition of b-NADPH2 to the wells initiates the progressive reduction of DTNB by GSH, causing a color increase that is monitored at 405 nm. The rate of color change, followed over a 5 minute time period, is proportional to the tGSH concentration. Consequently, the concentration of tGSH in unknown samples may be determined by reference to the standard curve. GSH reacts with DTNB to produce a colored ion, which absorbs light at 405 nm, and a mixed disulphide. This disulphide reacts with further quantities of GSH present to liberate another ion and GSSG. GSSG is reduced enzymatically to GSH which then re-enters the cycle (see figure below). Since GSSG represents only a small percentage of total acid-solution free glutathione, the resulting values for tGSH (which encompasses both GSH and GSSG) are expressed in units of GSH equivalents.

  1. Add 50 µL of each GSH standard or diluted or undiluted sample extract to each well.
  2. Add 50 µL each of DTNB and Oxidoreductase solutions to each well.
  3. Incubate the plate for 10 minutes at room temperature.
  4. Add 50 µL of b-NADPH2 solution to each well to start the reaction
  5. Within one minute after the addition of b-NADPH2, read the plate using a kinetic program that can monitor the reaction at one minute intervals for 10 minutes. The rate of color change is monitored in the 405 – 412 nm range (depending on the filter wavelengths available on your plate reader). Note: If your plate reader does not have a kinetic reading function, then read the plate and record the OD value at 0 minutes and again at 10 minutes.

Package Inserts

Product Documents

Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.

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