Glutathione (GSH) Assay Kit 192 Test Version


NWK-GSH01

$395.00 each



The NWLSS™ Glutathione Assay kit is a kinetic, rate based assay for quantification of total GSH (reduced and oxidized) in a variety of samples including blood, plasma, tissues and cultured cells. This product is also suitable for measurement of oxidized GSH (GSSG) after scavenging of reduced GSH.

Recommended for researchers wishing to assay GSH or GSSG in tissue homogenates, cell lysates & other biological fluid where GSH is present.

Introduction

Glutathione (GSH, g-glutamylcysteinylglycine), the primary non-protein sulfhydryl in aerobic organisms is synthesized in most cells. The ubiquitous tripeptide is formed by the combination of glutamic acid and cysteine, catalyzed by g-glutamylcysteinyl synthetase. Glycine is then added by glutathione synthetase to form GSH.

In addition to donating an electron during the reduction of hydroperoxides to the respective alcohols (or water in the case of hydrogen peroxide), GSH functions as a co-substrate in the metabolism of xenobiotics catalyzed by glutathione S-transferases. It is also a co-factor for several metabolic enzymes is involved in intracellular transport, functions as an antioxidant and radioprotectant and facilitates protein folding and degradation.

Test Principle

The NWLSS™ Glutathione Assay is a modification of the method first described by Tietze. The general thiol reagent, 5-5'-dithiobis[2-nitrobenzoic acid] (DTNB, Ellman's Reagent) reacts with GSH to form the 412 nm chromophore, 5-thionitrobenzoic acid (TNB) and GS-TNB. The GS-TNB is subsequently reduced by glutathione reductase and β-nicotinamide adenine dinucleotide phosphate (NADPH), releasing a second TMB molecule and recycling the GSH; thus amplifying the response. Any GSSG initially present in the reaction mixture or formed from the mixed disulfide reaction of GSH with GS-TNB is rapidly reduced to GSH and detectable in the assay.

Catalog Number: NWK-GSH01
Format: 192 tests presented as 2 X 96 well microtiter plate assays. Cuvette format for spectrophotometers also available.
Sample Requirements: Whole blood, plasma, tissues and cell lysates.
Sensitivity: 0.1 µM using standard method (for whole blood & some tissue types). 0.005 µM using modified high sensitivity method (for samples with lower GSH levels such as plasma or for detection of GSSG after reduced GSH has been scavenged). Contact NWLSS for details.
Intended Use: Quantitative measurement of total GSH (reduced plus oxidized form)
Storage and Stability: 12 months from date of manufacture if stored at 2-8C
Kit Contents: DTNB Reagent
NADPH
NADPH Diluent
GR Enzyme
GSH (GSSG) Calibrator
Assay Buffer

Answer: Regarding the early yellow color development: It's likely that the samples are too basic. This can be due to an error in neutralization of the MPA by NaOH.
Answer: We have not yet adapted our current assay for modern automated instruments but yes it is possible to adapt Tietze based GSH methodologies for use on automated instruments.
Answer: RBC’s should be fully disrupted after treatment with 5% MPA however, in the case of whole blood, some white cells may survive intact. Since the concentration of RBC GSH is so much higher than that of WBC’s it is unlikely that GSH lost to surviving WBC’s would change the data much for whole blood GSH. If it is concern we recommend low level sonication to make sure all white cells are properly disrupted.
Answer: Yes, Trichloroacetic Acid (TCA) can also be used. { Question: According to the product insert whole blood sample can be tested using product NWK-GSH01. But on Page 7 of the product insert it says: “Collect samples using EDTA, heparin, citrate or ACD anticoagulant." We are confused about this. Does "Whole blood" just mean "Plasma"?} Answer: No whole blood does not mean plasma but whole blood does need to be collected with anticoagulant present. If anticoagulant is not present the sample will clot and cease to be representative of whole blood (a non-clotted mixture of plasma, RBCs and white cells). Whole blood that is left to clot will yield serum and a big clot that is not acceptable for testing GSH.

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