Name: Glutathione Peroxidase Assay Kit (Colorimetric)
SKU: ab102530
Rating: 5 (2 reviews)

Glutathione Peroxidase Assay Kit (Colorimetric) ab102530 can be used to quantitate the activity of all of the glutathione dependent peroxidases present in samples of plasma, erythrocyte lysates, tissue homogenates, and cell lysates. Readout on any colorimetric (340 nm) plate reader.

- Cited in > 140 publications
- Individual kit components also available for purchase with a minimum order of 20 units. Contact us to discuss your needs.

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Biochemical assay - Glutathione Peroxidase Assay Kit (Colorimetric) (AB102530), expandable thumbnail

Publications

Key facts

Detection method: Colorimetric
Sample types: Urine, Plasma, Tissue Extracts, Cell culture supernatant, Serum, Platelets, Other biological fluids
Assay type: Enzyme activity
Reactive species: Mammals
Assay time: 40m
Sensitivity: = 0.5 mU/mL

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Target data

See full target information GPX1

Function

Catalyzes the reduction of hydroperoxides in a glutathione-dependent manner thus regulating cellular redox homeostasis. Can reduce small soluble hydroperoxides such as H2O2, cumene hydroperoxide and tert-butyl hydroperoxide, as well as several fatty acid-derived hydroperoxides. In platelets catalyzes the reduction of 12-hydroperoxyeicosatetraenoic acid, the primary product of the arachidonate 12-lipoxygenase pathway.

Alternative names

Glutathione peroxidase 1, GPx-1, GSHPx-1, Cellular glutathione peroxidase, Phospholipid-hydroperoxide glutathione peroxidase GPX1, GPX1

What's included?

2000 Test

Components

Active Glutathione Peroxidase

20 x 1 Vial

Active Glutathione Reductase

20 x 10 µL

Cumene Hydroperoxide

20 x 1 Vial

Glutathione

20 x 1 Vial

Glutathione Peroxidase Assay Buffer

20 x 50 mL

NADPH II

20 x 1 Vial

Key facts

Detection method: Colorimetric
Sample types: Urine, Plasma, Tissue Extracts, Cell culture supernatant, Serum, Platelets, Other biological fluids
Assay type: Enzyme activity
Reactive species: Mammals
Assay time: 40m
Assay Platform: Microplate reader
Sensitivity: = 0.5 mU/mL

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: -20°C
Appropriate long-term storage conditions: -20°C
Storage information: -20°C

Notes

How the assay works

Glutathione peroxidase assay kit measures the activity of glutathione peroxidase by measuring the decrease in absorbance of NADPH. The assay is based on the oxidation of reduced glutathione to oxidized glutathione.

In the glutathione peroxidase assay protocol, glutathione peroxidase (GPx) oxidizes GSH to produce GSSG as part of the reaction in which it reduces cumene hydroperoxide. Glutathione reductase (GR) then reduces the GSSG to produce GSH, and in the same reaction consumes NADPH. The decrease of NADPH (measured at OD=340 nm) is proportional to GPx activity.

Glutathione peroxidase assay protocol summary:

- Add samples and standards to wells
- Add reaction mix and incubate for 15 min at room temp to deplete all GSSG in sample
- Add cumene hydroperoxide
- Analyze with microplate reader immediately, and after at least 5 min

How other researchers are using

Glutathione Peroxidase Assay Kit (Colorimetric) has been used in a variety of sample type including:
Mouse C2C12 myoblasts ¹
Mice heart samples²
Rats gonads homogenates ³
References:

1-Martin S et al. 2023
2-Li H et al. 2023
3-Naguib G et al. 2023

Related and recommended products

For fluorometric detection, we recommend Glutathione Peroxidase Activity Assay Kit (Fluorometric) Glutathione Peroxidase Activity Assay Kit (Fluorometric) ab219926

This product is manufactured by BioVision, an Abcam company and was previously called K762 Glutathione Peroxidase Activity Colorimetric Assay Kit. K762-100 is the same size as the 100 test size of ab102530.

The Glutathione Peroxidase (GPx, EC 1.11.1.9) family of enzymes plays and important role in the protection of organisms from oxidative damage. GPx converts reduced glutathione (GSH) to oxidized glutathione (GSSG) while reducing lipid hydroperoxides to their corresponding alcohols or free hydrogen peroxide to water.

The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Glutathione Peroxidase (GPx) often referred to as GSH peroxidase is an essential enzyme in the cellular defense system against oxidative damage. It exists as a group of enzymes including the widely studied isoform GPx4. GPx has a molecular weight of approximately 20-23 kDa varying slightly depending on the isoform. This enzyme is localized in many tissues across different organs including the liver kidney and muscles. It catalyzes the reduction of hydrogen peroxide and organic hydroperoxides using glutathione (GSH) as a substrate preventing the formation of free radicals.

Biological function summary

The enzyme plays a significant role in the detoxification of reactive oxygen species. GPx does not usually form a complex with other proteins; instead it acts individually within cells to maintain the balance of oxidative and reductive forces. By converting harmful peroxides into non-toxic alcohols and water it protects cell membranes and other cellular components from oxidative damage. GPx activity is often measured in laboratories using assays to determine its efficiency in catalyzing these reactions.

Pathways

GPx engages in critical cellular antioxidant defense mechanisms. It is an integral part of the glutathione redox cycle where it partners with glutathione reductase another enzyme important for regenerating reduced glutathione. Additionally GPx links to the pentose phosphate pathway providing reduced NADPH necessary for maintaining glutathione in its reduced form. These pathways protect cells from oxidative stress and keep redox homeostasis in check.

Associated diseases and disorders

Alterations in GPx activity correlate with various health conditions. Researchers link decreased GPx activity to cardiovascular diseases and neurodegenerative disorders highlighting its importance in maintaining cellular health. In cardiovascular diseases the imbalance of oxidative stress pathways involves proteins like superoxide dismutase in addition to GPx worsening the effects of oxidative damage. In neurodegenerative disorders abnormal protein aggregations disturb GPx activities emphasizing the role of antioxidants in disease prevention and management.

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7 product images

Biochemical assay - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
Pesti-Asboth et al used Lipid Peroxidation (MDA) Assay Kit Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970, Vitamin C Assay Kit Ascorbic Acid Assay Kit (Biological Samples) ab65656, Glutathione Peroxidase Assay Kit ab102530, Glutathione Reductase Assay Kit Glutathione Reductase (GR) Assay Kit ab83461, and Superoxide Dismutase Assay Kit Superoxide Dismutase Activity Assay Kit (Colorimetric) ab65354 to investigate changes in redox in the plasma of fast growing broiler chickens.
Changes in the redox parameters in blood plasma at Days 3, 8, 21, 32, and 42. Significant differences were determined by comparing the data from each time point with the data from the first time point. Data are expressed as the means ±8 SEMs; *P<0.05, ** p<0.005, *** p<0.001.
Lipid peroxidation was determined using a commercially available assay kit (Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970, Abcam, Cambridge, United Kingdom). The measurements were based on the reaction between MDA and thiobarbituric acid (TBA). All reagents and standard solutions were prepared according to the manufacturer’s instructions. Plasma samples (20 μl) were mixed with 500 μL of 42 mM H2SO4, 125 μL of phosphotungstic acid solution was added, and each sample was mixed by vortexing and incubated at room temperature for 5 minutes. After incubation, we centrifuged the samples at 13000 × g for 3 minutes. The pellet was collected and resuspended in 100 μL of distilled water on ice with 2 μL of butylated hydroxytoluene (BHT) (100-fold dilution). The final volume was adjusted to 200 μL with distilled water. After sample preparation, the assay was performed. The MDA-TBA adduct was generated in each sample and standard solution. The TBA reagents (600 μl) were added to the samples and standards for incubation at 95°C for 60 minutes. The reaction mixes were cooled in an ice bath for 10 minutes after incubation. The samples and standards were placed in duplicate in a 96-well microplate for analysis. The absorbance of the MDA-TBA adduct was measured at 532 nm, and the calculated concentrations are expressed in nmol/ml.
To determine the vitamin C concentration, a plasma-specific assay kit was used (Ascorbic Acid Assay Kit (Biological Samples) ab65656, Abcam, Cambridge, United Kingdom) [34–36]. The measurements were performed according to the protocol in the manual with some modifications. The kit’s supernatant assay buffer was set to pH 7.0 with NaOH (10 M). The kit provides a sensitive method for vitamin C measurement. Plasma vitamin C reduces Fe3+ to Fe2+, which shows strong absorbance that can be monitored between 545–600 nm. The reagent and standard were prepared as described in the protocol. Fifty microliters of each plasma sample, run in duplicate, was diluted two fold for measurement. The assay procedure was followed as described in the manual. The absorbance of each sample was measured at 593 nm, and the concentrations were calculated as recommended and are expressed in nmol/ml.
The activity of Glutathione peroxidase (GPx) was measured with a commercially available kit (ab102530, Abcam, Cambridge, United Kingdom). In the assay, GSSG produced after GPx oxidizes GSH during H2O2 reduction. GSSG is reduced back to GSH by GR with the aid of nicotinamide adenine dinucleotide phosphate (NADPH). The reduction of NADPH is proportional to GPx activity and can be measured colorimetrically at 340 nm. The kit reagents were dissolved as described in the manual. A standard curve was prepared as described in the kit. The activity of GPx is expressed as mU/ml.
The activity of Glutathione reductase (GR) in plasma was determined using a specific assay kit (Glutathione Reductase (GR) Assay Kit ab83461, Abcam, Cambridge, United Kingdom). In this assay, GSH is formed from GSSG by GR; then, GSH reacts with DTNB, and a 2-nitro-5-thiobenzoate anion (TNB2-) is generated. The change in absorbance was measured at 405 nm. The kit reagents were dissolved as described in the “components and storage” section. The protocol recommends pretreating the samples. First, 5 μL of 3% H2O2 was added to 100 μL of each sample. The samples were incubated at 25°C for 5 min. Then, 5 μL of catalase was added to each sample, and we incubated the samples again at 25°C for another 5 min. After the pretreatment procedure, 50 μL of each pretreated sample was added to the sample wells. The standard curve was prepared as described in the manual, and 50 μL of diluted standard solution was added to each well. GR activity is expressed in nmol/min/mL = mU/ml.
The superoxide dismutase(SOD)inhibition rate was measured with a specific assay kit (Superoxide Dismutase Activity Assay Kit (Colorimetric) ab65354, Abcam, Cambridge, United Kingdom). In this assay, xanthine oxidase produced superoxide anions, and the conversion of superoxide anions into hydrogen peroxide was catalyzed by SOD. Superoxide anions and the water-soluble tetrazolium salt WST-1 can react to produce a water-soluble formazan dye, the absorbance of which was detected at 450 nm. The reagents were prepared as described in the kit. First, 20 μL of Blank 1, Blank 2, Blank 3 or sample was added to a 96-well plate in duplicate. Then, WST-1 solution (200 μl) was added to each blank and sample. Twenty microliters of dilution buffer were added to the Blank 2 and Blank 3 solutions. Enzyme working solution (20 μl) was added to each sample and Blank. Then, the plate was incubated at 37°C for 20 minutes. The absorbance (A) was then measured at 450 nm.
George, Akash K et al., Scientific reports: vol. 8,1 5158., Fig 4, doi:10.1038/s41598-018-23568-z
Functional Studies - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
Scatter dot plots representing the levels of glutathione peroxidase (GPx) in brain tissue in different mice groups.
Liu, Baohai et al., Frontiers in pharmacology: vol. 9 347., Fig 8, doi:10.3389/fphar.2018.00347
Functional Studies - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
The effects of PCSE on the activity of GPX in a mouse UC model.
Functional Studies - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
Standard curve: mean of duplicates (+/- SD) with background reads subtracted
Functional Studies - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
Glutathione Peroxidase Activity measured in biological fluids showing quantity (nmol) per 1 mL after 10 min of incubation. Samples diluted 1-27 fold.
Functional Studies - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
Glutathione Peroxidase Activity measured in cell lysates showing quantity (nmol) per 1 mln cells after 10 min of incubation. Undiluted sample contained 2.5e6 cells per well. Samples were diluted 1-9 fold.
Functional Studies - Glutathione Peroxidase Assay Kit (Colorimetric) (ab102530)
Tests example obtained using ab102530