Glutathione is a key intracellular tripeptide thiol composed of glutamic acid, cysteine, and glycine. Glutathione helps protect cells from free radical damage by acting as an antioxidant. Within cells, glutathione exists in reduced (GSH) and oxidized (GSSG) states. In red blood cells, the reduced form of glutathione is vital in maintaining hemoglobin in a reduced state and hence protecting the cells from oxidative damage. Glutathione is involved in detoxification of hydrogen peroxide through glutathione oxidase. Low levels of glutathione are found in deficiencies of key enzymes involved in glutathione metabolism, such as glucose-6-phosphate dehydrogenase, glutathione synthase and glutathione reductase.

Total Glutathione (GSSG/GSH) Assay Kit provides a simple, reproducible, and sensitive tool for measuring total glutathione (both GSH and GSSG) concentration in plasma, serum, cell lysates, urine, and other biological liquid samples. This assay employs an enzymatic recycling method that combines multiple steps. Initially, Glutathione Reductase reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH. Subsequently, the Colorimetric probe reacts with the thiol group of GSH to produce a colored compound that absorbs at 405 nm. The optical density measured at 405 nm is directly proportional to the concentration of total glutathione present in the sample.

• Microplate reader capable of measuring absorbance at 405 nm
• 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
• Ultrapure water
• 50 mL conical tubes
• Protocatechuic acid (HClO₄) (PCA)
• Potassium hydroxide (KOH)

1. Add 50 µL of the diluted GSSG Standards or unknown samples to the 96-well microtiter plate.
2. Add 50 µL of Reaction Mix to each well. Mix thoroughly.
3. Ensure that the plate reader is prepared for a kinetic assay and is set to read at 405 nm if using Kinetic method.
4. Add 50 µL of Colorimetric Solution to each well and mix briefly.
5. Total glutathione (GSSG/GSH) concentration of the samples can be determined by End point method or Kinetic method.