Cellular Glutathione Peroxidase Assay Kit with NADPH
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Cat. No.: CGPAK-100 (for 100T)
Cellular Glutathione Peroxidase Assay Kit with NADPH is a simple and convenient reagent kit used to detect the activity of Glutathione peroxidase (GPx) in cells, tissues, or other samples through UV colorimetry.
The majority of cellular Glutathione peroxidase enzymes contain selenium, which is an essential component of their active center. There are also small amounts of non-selenium-containing Glutathione peroxidase enzymes present in cells. This assay kit detects the most common selenium-containing Glutathione peroxidase.
The assay kit offers flexible and convenient usage, with a wide range of sample volumes and detection times. The sample volume can be adjusted between 0.1 and 50 microliters based on the activity of GPx in the sample, and the detection time can be adjusted between 5 and 30 minutes based on the GPx activity in the sample.
When used in conjunction with Total Glutathione Peroxidase Assay Kit with NADPH, this assay kit can quantitatively detect non-selenium-containing Glutathione peroxidase in the sample.
Glutathione peroxidase plays a critical role in protecting cells from oxidative damage by removing peroxides within living cells. Lipids in cells are susceptible to reaction with free radicals, leading to lipid peroxidation. Glutathione peroxidase utilizes reduced glutathione (GSH) to reduce lipid peroxides, thereby eliminating the toxic effects of free radicals. Glutathione peroxidase is found in almost all tissues. In certain pathological conditions, the activity of Glutathione peroxidase can be significantly upregulated or downregulated.
Glutathione peroxidase can catalyze hydrogen peroxide and many organic peroxides using reduced glutathione (GSH), resulting in the production of water or organic alcohols. However, detecting hydrogen peroxide as a substrate can be affected by catalase, which is an enzyme that can also decompose hydrogen peroxide. Catalase activity can interfere with the measurement of Glutathione peroxidase. This assay kit utilizes an indirect measurement method. Glutathione peroxidase (GPx) catalyzes the production of oxidized glutathione (GSSG) from GSH, while glutathione reductase uses NADPH to catalyze the production of GSH from GSSG. The decrease in NADPH can be measured at A340, allowing for the calculation of Glutathione peroxidase activity. In this reaction, Glutathione peroxidase is the rate-limiting step of the entire reaction system, so the reduction of NADPH is linearly correlated with the activity of Glutathione peroxidase.
The provided organic peroxide reagent (t-Bu-OOH) in this assay kit does not react with GSH in the absence of Glutathione peroxidase and is not decomposed by intracellular catalase. Therefore, it can selectively detect the activity of Glutathione peroxidase.
Since t-Bu-OOH is not catalyzed by non-selenium-containing Glutathione peroxidase, this assay kit can quantitatively detect the most common selenium-containing Glutathione peroxidase with higher specificity.
The assay kit can detect the activity of Glutathione peroxidase in samples such as tissue homogenates and cell lysates. One kit allows for 100 tests.
- This assay kit involves redox reactions, so all oxidizing agents or reducing agents will interfere with the assay. If reducing agents in the sample cannot be avoided, such as DTT, mercaptoethanol, etc., the total concentration of these reducing agents should be at least below 0.1 mM. DTT at a concentration of 0.15 mM can inhibit 40% of enzyme activity.
- Common detergents like Triton X-100 and Tween-20 contain high levels of peroxides, which can affect the assay. If it is necessary to use these detergents, it is recommended to use high-purity ones with lower levels of peroxides.
- The initial reading after adding the peroxide reagent solution, such as the A340 reading at 0 minutes, should not be lower than 1. If the reading is below 1, it indicates that the GPx activity in the sample is too high or the GSSG content in the sample itself is too high. In such cases, the sample needs to be appropriately diluted or the amount of the sample reduced. Otherwise, the reaction will quickly reach a plateau phase, and accurate measurement data cannot be obtained. The sample can be tested immediately or stored at -70°C for future testing.
- It is crucial to strictly control the temperature during the reaction at 25°C to minimize errors.
- NADPH is not very stable, so it is important to strictly follow the instructions to prevent its inactivation.
- This product is intended for scientific research purposes by professionals only. It should not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in residential areas.
- For your safety and health, please wear appropriate laboratory attire and disposable gloves during the operation.
Store at -20°C, valid for one year. After dissolving NADPH, it is recommended to aliquot and store at -70°C. It can be stored at 4°C for one day, but after one week at -20°C, NADPH may degrade by more than 10%. Once prepared as a solution, GSH should be aliquoted and stored at -20°C.
Only for research and not intended for treatment of humans or animals
SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory