Glutathione reductase is a homodimeric enzyme that belongs to the flavoprotein disulfide oxidoreductases family. It plays an indirect role in the prevention of oxidative damage in cells by maintaining intracellular reduced glutathione (GSH). Thus, measuring the activity of this enzyme is an indicator of oxidative stress. It is a ubiquitous enzyme that catalyzes the NADPH dependent reduction reaction of oxidized glutathione (GSSG) to reduced glutathione (GSH). Maintaining GSH is vital for oxidation-reduction processes and for detoxifying hydrogen peroxide and organic peroxides produced by inflammation in cells.

Glutathione Reductase (GR) Activity Assay Kit offers a user-friendly, reliable, and highly sensitive method for quantifying GR activity in plasma, serum, cell lysates, and other biological fluid samples. In this assay, Glutathione Reductase reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340nm. The rate of decrease in the absorbance is directly proportional to the GR 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 NADPH Solution.
4. Add 20 µL of the diluted GR Positive Control, Assay Buffer (used as a blank) or unknown samples to a 96-well microtiter plate.
5. Add 100 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 40 µL of the NADPH Solution into each well (it is recommended to use a multi-channel pipette) and mix thoroughly.
7. Read and record absorbance at 340 nm every minute for 5 time points at 25°C.