Description
Key facts
Detection method:Fluorescent,
Sample types:Urine, Plasma, Tissue Extracts, Serum, Cell Lysate,
Assay type:Quantitative,
Sensitivity:= 10 nM,
Assay time:30m,
Assay Platform:Microplate reader
Product details:
How the assay works
The GSH/GSSG assay protocol uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with GSH. With a one-step fluorimetric method, the assay can detect as little as 1 picomole of GSH or GSSG in a 100 µL assay volume.
Glutathione (GSH) is a tripeptide that contains L-cysteine, L-glutamic acid and glycine. It is the smallest intracellular protein thiol molecule in the cells, which prevents cell damage caused by reactive oxygen species such as free radicals and peroxides. Glutathione exists in reduced (GSH) and oxidized (GSSG) states.
Reduced glutathione (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 generates oxidized glutathione (GSSG).
Glutathione reductase (GR) recycles GSSG to GSH with the simultaneous oxidation of ß-nicotinamide adenine dinucleotide phosphate (ß-NADPH2).
In healthy cells, >90% of the total glutathione pool is in the reduced form (GSH). When cells are exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSSG to GSH increases. An increased ratio of GSSG-to-GSH is an indication of oxidative stress.
The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation without a separation step. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm.
GSH/GSSG assay protocol summary:
- add samples (deproteinized) and standards to wells
- for GSH assay add Thiol Green in assay buffer, or for total glutathione (GSH + GSSG) assay also add GSSG probe
- incubate for 10 - 60 min at room temp
- analyze with microplate reader
GSSG levels can be calculated by subtracting GSH from total glutathione levels.
NOTE: For measuring GSH Standard only, there is enough reagent provided to perform 200 tests.
Alternative assay kits
As an alternative to this kit, we recommend GSH/GSSG Ratio Detection Assay Kit II (Fluorometric - Green) (ab205811) that is a reformulated version of ab138881 with similar performance. It uses a water-soluble probe, which is more stable, than the DMSO-soluble dye used in ab138881.
How other researchers are using
GSH/GSSG Ratio Detection Assay Kit has been used in a variety of sample type including:
- Human retinal microvascular endothelial cells 1
- Rat cardiomyocyte-derived H9C2 cells 2
- Murine B16 melanoma, human MCF-7 breast cancer, SW1990 pancreatic carcinoma cell lines 3
References:
1 - Niu T et al. 2024; 2 - Li D et al. 2024; 3 - Zhou N at al. 2023.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
