Glutathione Peroxidase (GSH-Px) Activity Assay Kit

Glutathione Peroxidase (GSH-Px) Activity Assay Kit

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N V Mohamad et al investigate the therapeutic effect of annatto tocotrienol for postmenopausal bone loss. Glutathione peroxidase (GSH-Px) activity of rat serum was determined using GSH-Px activity assay kit (E-BC-K096-S).

A significant decline of GSH-Px activity due to ovariectomy (OVX) was observed (P=0.039). Treatment with annatto tocotrienol (ANTT) and Raloxifene (RAL) elevated GSH-Px activity.

Dilution of Sample

The optimal sampling volume are different for different species, also are different for different sample type. It is recommended to take 2~3 samples to do a pre-experiment, diluting a series of diluent and determine the dilution factor when the inhibition ratio is 20%~60% (the optimal inhibition ratio is the range of 45%~55%.) before formal experiment.

Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).

Detection Principle

Glutathione peroxidase (GSH-Px) can promote the reaction of hydrogen peroxide (H2O2) and reduced glutathione to produce H2O and oxidized glutathione (GSSG). The activity of glutathione peroxidase can be expressed by the rate of enzymatic reaction. The activity of glutathione can be calculated by measuring the consumption of reduced glutathione. Hydrogen peroxide (H2O2) and reduced glutathione can react without catalysis of GSH-Px, so the portion of GSH reduction by non-enzymatic reaction should be subtracted. GSH can react with dinitrobenzoic acid to produce 5-thio-dinitrobenzoic acid anion, which showed a stable yellow color. Measure the absorbance at 412nm, and calculate the amount of GSH.

Kit Components & Storage

Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.

Technical Data:

Parameter:

Intra-assay Precision

Three human serum samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).

Inter-assay Precision

Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.

Recovery

Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 105%.

Sensitivity

The analytical sensitivity of the assay is 12.65 U. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.

Standard Curve

As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only: