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Glutathione peroxidase (GPX) is a tetramer composed of four identical subunits. It requires selenium as a cofactor and contains a selenocysteine amino acid residue in the active site of each monomer that participates in the actual mechanism of the enzyme. Glutathione peroxidase belongs to an enzyme family whose function is to detoxify peroxides in the cell. Because peroxides can decompose to form highly reactive radicals, the GPX enzymes play a critical role in protecting the cell from free radical damage, particularly lipid peroxidation.

Glutathione Peroxidase (GPX) Activity Assay Kit offers a user-friendly, reliable, and highly sensitive method for quantifying GPX activity in plasma, serum, cell lysates, and other biological fluid samples. In this assay, GPX reduces hydroperoxides, producing oxidized glutathione (GSSG), which is then recycled back to its reduced state by glutathione reductase (GR) and NADPH. The reduction of GSSG to GSH is accompanied by the oxidation of NADPH to NADP+, leading to a decrease in absorbance at 340nm. The rate of decrease in the absorbance is directly proportional to the GPX activity in the sample.

• Microplate reader capable of measuring absorbance at 340 nm at 25°C
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Multi-channel pipettes to deliver 20 μL to 200 μL volumes
• Tubes to prepare sample dilutions
• 15 mL conical tubes
• Incubator at 25°C
• Deionized water

1. Prepare Working Solution.
2. Prepare Positive Control.
3. Prepare Cumene Hydroperoxide Solution.
4. Add 20 μL of the diluted GPX positive control, Assay Buffer (served as a blank), or sample to each well of a 96-well microtiter plate.
5. Add 160 μL of Working Solution to each well. Mix thoroughly. Incubate for 5 min at 25°C with gentle shaking.
6. After five minutes, initiate the reaction by adding 20 μL of the Cumene Hydroperoxide Solution into each well and mix thoroughly (it is recommended to use a multi-channel pipette).
7. Immediately after, read and record absorbance at 340nm every minute for 5 time points at 25°C.