Glutahione Peroxidase Assay Kit

Catalog No. ABIN1000313

Product Details

Target: Glutahione Peroxidase

Application: Activity Assay (AcA)

Specificity: 12 U/L

Characteristics: Sensitive and accurate. Use 10 µL sample. Linear detection range 12 to 300 U/L GPX activity.

Components: Assay Buffer: 25 mL. GR Enzyme: 1 mL. Glutathione: 240 µL. NADPH: 40 µL. H2O2 Solution: 100 µL. Calibrator: 100 µL Positive Control: 9 µL Glutathione Peroxidase (GPX).

Material not included: Pipetting devices, centrifuge tubes, clear flat-bottom uncoated 96-well plates, plate reader capable of reading optical density at 340nm every minute, homogenizer etc.

Application Details

Application Notes: Direct Assays: GPX activity in biological samples.
Drug Discovery/Pharmacology: effects of drugs on GPX activity.

Protocol: 1. Standards and Samples. Mix 12 µL of the Calibrator with 188 µL dH2O (equivalent to 6 mM NADPH). Dilute this calibrator stock as shown in the Table below. Transfer 10 µL standards into wells of a clear flat-bottom 96-well plate. Add 190 µL Assay Buffer to all standard wells. Transfer 10 µL sample and 10 µL reconstituted GPX Positive Control into separate wells of the 96-well plate. In addition, for each assay run, include a background control that only contains 10 µL Assay Buffer. Note: (1). For unknown samples, perform several dilutions to ensure that GPX activity is within the linear range of 12 to 300 U/L. (2) The provided GPX serves as a positive control to ensure assay is working and should not be used to calculate the Sample GPX activity.
2. Assay. Prepare enough Working Reagent for Sample and Control wells by mixing, for each well, 85 µL Assay Buffer, 2 µL Glutathione, 2 µL 35 mM NADPH and 8 µL GR enzyme. Add 90 µL Working Reagent quickly to the Sample/Control wells. Tap plate to mix. Dilute 8 µL 3% H2O2 with 1992 µL dH2O (final
3.5 mM). Prepare enough 0.35 mM H2O2 Reagent by mixing, for each Sample/Control well, 12 µL
3.5 mM with 108 µL dH2O. Use this Reagent within one hour. With a multi-channel pipettor, add 100 µL 0.35 mM H2O2 Reagent to all Sample and Control wells. Tap plate quickly to mix well contents thoroughly. Immediately read OD340nm (time zero, OD0) and again at 4 min (OD4).

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Chen, Cheng, Chen, Yi, Nie, Sun, Qin, Tian, Jin, Zhang: "Lycium barbarum polysaccharides prevent memory and neurogenesis impairments in scopolamine-treated rats." in: PLoS ONE, Vol. 9, Issue 2, pp. e88076, (2014) (PubMed).

Nakyinsige, Man, Aghwan, Zulkifli, Goh, Abu Bakar, Al-Kahtani, Sazili: "Stunning and animal welfare from Islamic and scientific perspectives." in: Meat science, Vol. 95, Issue 2, pp. 352-61, (2013) (PubMed).

Calamita, Gena, Ferri, Rosito, Rojek, Nielsen, Marinelli, Frühbeck, Svelto: "Biophysical assessment of aquaporin-9 as principal facilitative pathway in mouse liver import of glucogenetic glycerol." in: Biology of the cell / under the auspices of the European Cell Biology Organization, Vol. 104, Issue 6, pp. 342-51, (2012) (PubMed).

Target Details

Target: Glutahione Peroxidase

Background: Quantitative determination of glutathione peroxidase activity at 340nm.
Procedure: 20 min.

Glutathione Peroxidase (GPX, EC 1.11.1.9) represents an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. It helps prevent lipid peroxidation of cellular membranes by removing free peroxide in the cell. Simple, direct and high-throughput assays for GPX activity find wide applications. This improved assay directly measures NADPH consumption in the enzyme coupled reactions. The measured decrease in optical density at 340nm is directly proportional to the enzyme activity in the sample.