Glutathione (GSH) is a tripeptide comprised of three amino acids L-cysteine, L-glutamic acid and glycine. It is involved in the development and maintenance of protein disulfide bonds, the transport of amino acids across cell membranes, and in detoxification. Since glutathione contains a thiol group, it serves as a major endogenous antioxidants in cells preventing damage by neutralizing reactive oxygen species
such as free radicals and peroxides. Monitoring reduced and oxidized glutathione in biological samples is a useful tool for evaluating the redox and detoxification status of cells and tissues against oxidative and free radical mediated cell injury. While few reagents and assay kits are commercially available for quantifying glutathione, they are hindered by their lack of sensitivity and tedious protocols. Amplite™ Fluorimetric Glutathione assay offers a convenient and ultra-sensitive method for the quantification of glutathione in biological samples.
GSH dose responses were measured with Amplite™ Fluorimetric Glutathione Assay Kit (Cat# 10055
) on a 96-well black solid plate. As low as 10 nM (1 pmol/well) GSH was detected with 10 minutes incubation.
Amplite™ Fluorimetric Glutathione Assay
Fluorimetric Glutathione assay is a sensitive, one-step fluorimetric method to detect as little as 1 picomole of GSH in a 100 µL assay volume. This assay employs a proprietary non-fluorescent and pH-dependent glutathione sensor that becomes strongly fluorescent upon reacting with thiol. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and is easily adaptable for automation without a separation step. The fluorescence signal can be easily detected using a fluorescence microplate reader at Ex/Em = 490/520 nm.
Glutathione GSH/GSSG Ratio
In healthy cells and tissues, glutathione exists in two states, reduced (GSH) and oxidized (GSSG). In the reduced state, GSH is a major tissue antioxidant that neutralizes reactive oxygen species. As a result of donating an electron, GSH itself becomes reactive and will readily react with other reactive GSH molecules to form GSSG. Additionally, reduced GSH 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 forms GSSG. The enzyme glutathione reductase (GR) recycles GSSG to GSH while simultaneously oxidizing β-nicotinamide adenine dinucleotide phosphate (β-NADPH2).
Monitoring reduced GSH and its ratio with oxidized GSSG may be used to evaluate the redox and detoxification status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury. Under normal conditions, the GSH redox couple in mammalian cells typically ranges in concentration from 1 - 10 nM, with resting cells having molar GSH:GSSG ratios exceeding 100:1. When cells are exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSH/GSSG decreases to ratios as low as 10:1 and even 1:1. As such, monitoring GSH/GSSG levels in biological samples has become a useful tool in evaluating oxidative stress of cells and tissues, and cell injuries caused by free radicals.
GSH and Total (GSH + GSSG) dose responses were measured with Amplite™ Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit. Blue line: in the presence of GSH only. Red line: in the presence of 1:1 GSH/GSSG.
Amplite™ Glutathione GSH/GSSG Ratio Assays
AAT Bioquest Amplite™ Fluorimetric Glutathione GSH/GSSG Ratio Assay
and Amplite™ Rapid Fluorimetric Glutathione GSH/GSSG Ratio Assay
kits are simple and ultrasensitive assays to quantify as little as 1 picomole of GSH (or cysteine) in a 100 µL assay volume. Each kit uses a proprietary non-fluorescent sensor that becomes strongly fluorescent upon reacting with GSH, and its signal can be detected using a fluorescence microplate reader at Ex/Em = 490/520 nm. Assays can be performed in a convenient 96-well or 384-well microtiter-plate format and is easily adaptable for automation without a separation step. What differs between these assays is the sensor's solubility. Amplite™ Fluorimetric Glutathione GSH/GSSG Ratio assay uses sensor Thiolite™ Green which is soluble in DMSO. Amplite™ Rapid Fluorimetric Glutathione GSH/GSSG Ratio assay uses a water-soluble sensor Thiolite™ Green 520WS.