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Glutathione Metabolism

Glutathione (GSH), an intracellular low-molecular-weight thiol, is a key antioxidant in many species  and plays a critical role in cellular defense against oxidative stress in tissues and cells. It has been highly implicated in the detoxification/elimination of xenobiotics (naturally occurring harmful compounds such as free radicals, hydroperoxides etc.) and in the maintenance of the oxidation state of protein sulfhydryl groups. In addition, GSH plays a pivotal role in the pathogenesis of numerous human disorders including cancer and cardiovascular diseases as well as in inflammation and aging processes.
Glutathione is present in cells in both reduced (GSH) and oxidized (GSSG) forms. Under normal physiological conditions, GSH is the predominant form inside cells. Thus, pathologic conditions causing oxidative stress would result in increased levels of GSSG and therefore, the measurement of intracellular GSH appears to be a sensitive indicator of the overall cell health, and its ability to resist toxic challenges.

Glutathione Detection Kits

The Colorimetric Glutathione Detection Kit provides a convenient, colorimetric method for analyzing either total glutathione or only the reduced form of glutathione using a microtiter plate reader. The assay utilizes the glutathione recycling system with DTNB and glutathione reductase: GSH is regenerated from GSSG by glutathione reductase, and will again react with DTNB to produce yellow 2-nitro-5-thiobenzoic acid that can be determined spectrophotometrically at 412 nm. This recycling reaction dramatically improves the sensitivity of total glutathione detection - the kit can quantify total glutathione from 1-100 ng/well. For detecting lower glutathione concentrations, such as in blood samples, increasing reaction time will significantly improve the signal. The kit can also specifically detect the reduced form of glutathione (GSH) by omitting the glutathione reductase from the reaction mixture.

Diminished glutathione (GSH) levels occur at the early stages of apoptosis. The PromoKine Fluorometric Glutathione Detection Kit provides a simple in vitro assay for detecting the GSH changes in apoptosis and other pathological processes. The assay utilizes a cell-permeable dye (monochlorobimane, MCB) that has a high affinity for glutathione. The unbound dye is almost nonfluorescent; whereas the dye fluoresces intensively blue (excitation = 380 nm; emission = 461 nm) when bound to reduced or oxidized glutathione (GSH-MCB). The reaction is catalyzed by glutathione S-transferase (GST). Thus, the decrease in glutathione level (e.g. in apoptotic cells) can be easily detected using a fluorometer or a 96-well fluorometric plate reader.

PromoKine's Intracellular Glutathione (GSH) Detection Kit also utilizes monochlorobimane (MCB), and provides a useful tool for fast and easy evaluation of GSH effectors directly in the living cells.

Reduced Glutathione (GSH) Assay Kits

PromoKine’s Reduced Glutathione Assay Kit I provides a simple and reproducible colorimetric method to specifically detect as low as 50 pmol/well of the reduced form of Glutathione (GSH) in a 100 µl reaction. The assay is based on an enzymatic cycling method in the presence of GSH and a chromophore. The reduction of the chromophore produces a stable product, which can be followed kinetically at 450 nm. Therefore, its absorbance is directly proportional to the amount of GSH in the sample.   
Our Reduced Glutathione Assay Kit II is based on a specific enzymatic cycling method in the presence of GSH and a fluorophore. The reduction of the fluorophore produces a stable fluorescent product, the fluorescence of which is directly proportional to the amount of GSH in the sample and can be followed kinetically (Ex/Em=535/587 nm). The assay is simple, reproducible and can specifically detect as low as 8 pmol/well of reduced form of Glutathione (GSH) in a 100 µl reaction.
Our kits are the most specific and sensitive quantitative assays in the market because GSSG does not interfere with the assay. The kits include 5-sulfosalicylic acid (SSA) in order to protect samples’ GSH endogenous content (SSA acts as a deproteinizing and antioxidant agent).

Glutathione Reductase Activity Kit

Glutathione Reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), which plays an important role in the GSH redox cycle that maintains adequate levels of reduced GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress.

PromoKine’s Colorimetric Glutathione Reductase Activity Kit has been designed for measuring glutathione reductase (GR) activity in biological samples. It is highly sensitive and well suited for high-throughput. The assay is based on the GR-catalysed reduction of GSSG to GSH, which reacts with 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) to generate the yellow TNB2-. The kit can detect 0.1 – 40 mU/ml GR in various samples.

GST Assay Kits

Glutathione S-transferase (GST) is an essential enzyme involved in detoxification of xenobiotics mediating conjugation of glutathione with those compounds.
The Fluorometric and Colorimetric GST Assays can detect GST activity in crude cell lysate or purified protein fractions and the kits also suited for quantitation of GST-tagged fusion proteins. They are based upon the GST-catalyzed reaction between GSH and the GST substrate CDNB (Colorimetric Kit) or MCB (Fluorometric Kit).
CDNB has the broadest range of isozyme detectability (e.g. α-, µ-, phi-, and other GST isoforms). Under certain conditions, the interaction between glutathione and CDNB is totally dependent on the presence of active GST. Detection limit is approximately 10 ng active GST/assay.
The free form of MCB is almost non-fluorescent, whereas the dye fluoresces blue (Ex/Em = 380/461 nm) when reacting with glutathione. GST catalyzes the MCB-glutathione reaction, and the fluorescence levels are proportional to the amount of GST present in the reaction. Therefore, the GST level in samples can be easily detected using a fluorometer or a 96-well fluorometric plate reader.

References, Questions & Answers

Product Name Description Size Catalog Number Information Prices
Colorimetric Glutathione Detection Kit Colorimetric Glutathione Detection Kit for detecting the GSH changes in apoptosis and other pathological processes 100 assays PK-CA577-K261 click to select country
Fluorometric Glutathione Detection Kit Glutathione Detection Kit for apoptosis and other pathological processes 100 assays PK-CA577-K251 click to select country
Intracellular Glutathione (GSH) Detection Kit This kit provides a simple, fluorometric method for detection of intracellular glutathione in mammalian cells 100 assays PK-CA577-K504 click to select country
Reduced Glutathione (GSH) Assay Kit I Fast and simple colorimetric assay for detecting reduced GSH levels in mammalian cells, while oxidized GSSG is not detected 100 assays PK-CA577-K464 click to select country
Reduced Glutathione (GSH) Assay Kit II Fast and simple fluorometric assay for detecting reduced GSH levels in mammalian cells, while oxidized GSSG is not detected 100 assays PK-CA577-K740 click to select country
Colorimetric Glutathione Reductase Activity Kit Colorimetric quantitation of glutathione reductase activity 200 assays PK-CA577-K761 click to select country
Colorimetric GST Assay Kit Detecting GST activity by spectrophotometer at 340 nm 100 assays PK-CA577-K263 click to select country
Fluorometric GST Assay Kit Detecting GST activity by fluorometer or fluorescence plate reader. 100 assays PK-CA577-K260 click to select country
GST Inhibitor-2 (ethacrynic acid) An effective inhibitor of Glutathione S-transferase (GST) 1 mg PK-CA577-1556-1 click to select country
GST protein (active) Active Glutathione S-transferase 1 mg PK-RP577-1243-1 click to select country
Oxidized Glutathione (GSSG) Hydrogen acceptor in the enzymatic determination of NADP and NADPH 1 g PK-CA577-1241-1 click to select country
Reduced Glutathione (GSH) A biological antioxidant that helps protect cells from reactive oxygen species 1 g PK-CA577-1242-1 click to select country