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Glutathione Peroxidase (GSH-Px) Activity Assay Kit
SKU: E-BC-K096-M-500
Glutathione Peroxidase (GSH-Px) Activity Assay Kit
| SKU # | E-BC-K096-M |
| Detection Instrument | Microplate reader (400-420 nm, optimum wavelength: 412 nm) |
| Detection Method | Colorimetric method |
Product Details
Properties
| Synonyms | GSH-PX |
| Sample Type | Serum, plasma, cells, cell culture supernatant, tissue |
| Sensitivity | 17.17 U |
| Detection Range | 17.17-518.32 U |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 40 min |
| Precision | Average inter-assay CV: 6.200% | Average intra-assay CV: 2.400% |
| Other instruments required | Micropipettor, Incubator, Vortex mixer, Centrifuge |
| Other reagents required | Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4) |
| Storage | 2-8℃ |
| Valid period | 12 months |
Images
JC Huang et al investigate the relationship between selenium-related gene ALAD and Keshan disease. Glutathione peroxidase (GSH-Px) activity of human serum was determined using GSH-Px activity assay kit (E-BC-K096-M).
The activity of GPx was significantly reduced in the serum of patients with Keshan disease. (**P<0.01)
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 10%~50% (the optimal inhibition ratio is the range of 25%~45%.) before formal experiment.
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| Human serum | 1 |
| Mouse serum | 3-5 |
| Rat serum | 5-8 |
| 10% Mouse brain tissue homogenate | 1 |
| 10% Rat liver tissue homogenate | 30-60 |
| HepG2 cell homogenate (5 mgprot/mL) | 1 |
| 10% Plant tissue homogenate | 1-5 |
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
| Item | Component | Size (96 T) | Storage |
| Reagent 1 | Stock Solution | 0.5 mL × 1 vial | 2-8℃, 12 months |
| Reagent 2 | Acid Reagent | 50 mL× 1 vial | 2-8℃, 12 months |
| Reagent 3 | Phosphate | 12 mL × 1 vial | 2-8℃, 12 months |
| Reagent 4 | DTNB Solution | 7 mL× 1 vial | 2-8℃, 12 months, shading light |
| Reagent 5 | GSH Standard | 3.07 mg × 1 vial | 2-8℃, 12 months |
| Reagent 6 | GSH Standard Stock Diluent | 1.5 mL × 2 vials | 2-8℃, 12 months |
| Microplate | 96 wells | No requirement | |
| Plate Sealer | 2 pieces |
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).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U) | 88.50 | 264.50 | 856.40 |
| %CV | 2.6 | 2.3 | 2.3 |
Inter-assay Precision
Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U) | 88.50 | 264.50 | 856.40 |
| %CV | 8.4 | 9.0 | 8.7 |
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 104%.
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc. (μmol/L) | 15.5 | 43.5 | 74.3 |
| Observed Conc. (μmol/L) | 15.7 | 46.1 | 78.0 |
| Recovery rate (%) | 101 | 106 | 105 |
Sensitivity
The analytical sensitivity of the assay is 34.34 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:
| Concentration (mg/mL) | 0 | 10 | 20 | 40 | 50 | 60 | 80 | 100 |
| Average OD | 0.068 | 0.109 | 0.151 | 0.232 | 0.274 | 0.316 | 0.400 | 0.482 |
| Absoluted OD | 0 | 0.041 | 0.083 | 0.164 | 0.206 | 0.248 | 0.332 | 0.414 |