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Product Details
| Cat # +Size | K762-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (340 nm) |
| Species Reactivity | Mammalian |
| Applications | The assay has a detection sensitivity ~ 0.5 mU/ml of GPx in samples. |
| Features & Benefits | • Simple procedure; takes ~ less than 40 minutes • Fast and convenient |
| Kit Components | • GPx Assay Buffer • NADPH (lyophilized) • Glutathione Reductase • Glutathione (GSH, lyophilized) • Cumene Hydroperoxide • GPx Positive Control (lyophilized) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Glutathione Peroxidase (GPx, EC 1.11.1.9) is an enzyme family with peroxidase activity, and plays important role in protecting of organisms from oxidative damage. It converts reduced glutathione (GSH) to oxidized glutathione (GSSG), to reduce lipid hydroperoxides to their corresponding alcohols, or reduce free hydrogen peroxide to water. Several isozymes have been found in different cellular locations and with different substrate specificity. Low levels of GPx have been correlated with free radical related disorders. BioVision’s Glutathione Peroxidase Assay Kit measures glutathione peroxidase (GPx) activity through a coupled reaction with glutathione reductase (GR). In the assay, GPx reduce Cumene Hydroperoxide, and oxidize GSH to GSSG. The generated GSSG is reduced to GSH with consumption of NADPH by GR. The decrease of NADPH is proportionally to GPx activity in the reactions. The decrease of NADPH can be easily measured by absorbance at 340 nm. The assay can be used to measure all of the glutathione dependent peroxidases in plasma, erythrocyte lysates, tissue homogenates, and cell lysates with detection sensitivity ~ 0.5 mU/ml of GPx in samples.
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What is the detection minimum of this kit?
The assay has a detection sensitivity of 0.5 mU/ml of glutathione peroxidase in samples.
Can this kit be used with plasma and whole blood?
The datasheet contains instructions for Erythrocytes. Whole blood can be processed similarly. Plasma can be diluted over a range and then the dilution that gives readings within the linear range of the standard curve can be used for the assay.
What is the activity level of the positive control? How can we increase its value to be comparable with our samples?
The positive control is only a benchmark sample. As long as the values are within the range of the std curve this is fine. The positive control is not be used to compare values with the samples. The positive control is provided to validate that the assay components are all working. The customer can add more volume to get higher values but this is not necessary as long as the values are within the std. curve range. GPx is a very vulnerable enzyme to freeze-thaw and can lose activity with storage over time.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples.
Can frozen samples be used with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Is it possible to use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
Why are the standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, the incubation time can be increased. As long as the standard curve is linear, it should be fine to use, since all the samples will also be measured under the same conditions on this curve.
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted.
Can RIPA buffer be used to prepare samples for this kit?
For any enzyme assay, we do not recommend RIPA buffer since it contains SDS and this can denature proteins and affect enzyme activity. We have tested and recommend using our Assay bufefr provided in the kit for best results.
Why do you add Cumene hydroperoxide to Reagent Control ? Will this anyway generate some signal ?
In this assay, Glutathione Peroxidase (GPx) reduces Cumene Hydroperoxide while oxidizing Glutathione (GSH) to oxidized glutathione (GSSG). The generated GSSG is reduced to GSH with consumption of NADPH by Glutathione reductase (GR). The decrease of NADPH (easily measured at 340 nm) is proportional to GPx activity. Reagent Control is included to determine if there is any reduction of Cumene Hydroperoxide that is happening in the absence of GPx. If so, then it needs to be subtracted from your Sample. Basically, Reagent Control will get rid of any non-specific signal that is generated so that we can guarantee that the signal generated is from GPx.
Zhou Zhou, Adipose‐Specific Lipin‐1 Overexpression Renders Hepatic Ferroptosis and Exacerbates Alcoholic Steatohepatitis in Mice. Hepatol Commun, May 2019; 31061954.
Long Chen, LncRNA GAS5 regulates redox balance and dysregulates the cell cycle and apoptosis in malignant melanoma cells. J Cancer Res Clin Oncol, Dec 2018; 30569211.
Jesmin Ara, et al., (2018) Hydrogen Water Drinking Exerts Antifatigue Effects in Chronic Forced Swimming Mice via Antioxidative and Anti-Inflammatory Activities, BioMed Research International, Apr. 2018, 29850492
Musabayane, C. T. et al., (2017) Kidney Function in P Berghei-Infected Sprague-Dawley Rats Following Treatment with Transdermally Delivered Syzygium-aromaticum Derived Oleanolic Acid, J Endocrinology and Thyroid Res, Apr.2017, 1(3)
Sibiya, HP et al., (2017) Kidney Function in P. Berghei-Infected Sprague-Dawley Rats Following Treatment with Transdermally Delivered Syzygium-aromaticum Derived Oleanolic Acid, J Endocrinol Thyroid Res, 2017, 1(3): 555565
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