Glycerol is an important metabolite found in all lipids and is essential for energy production, via glycolysis, and storage, via gluconeogensis. Excess levels of glycerol (hyperglycerolemia) usually is derived from glycerol kinase deficiency, which in turn leads to impaired energy regulation. This can trigger metabolic issues like insulin resistance, type 2 diabetes, and impaired glucose tolerance. Glycerol’s usage is extremely versatile and found to be used in pharmaceuticals, food industry, and industrially, etc. Monitoring glycerol levels is an important indication of lipid metabolism, cardiovascular diseases, and understanding energy regulation.

Free Glycerol Assay Kit provides a simple, reproducible, and sensitive tool of measuring glycerol concentration in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay employs a coupled enzymatic reaction system that combines multiple steps. Glycerol is oxidized with the conversion of the dye into a colored product that is measurable at 570 nm. The optical density measured at 570 nm is directly proportional to the concentration of glycerol present in the sample.

• Microplate reader capable of measuring absorbance at 570 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• Orbital shaker capable of 37°C
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the diluted Glycerol Standards or samples to the 96-well microtiter plate.
2. Initiate the reaction by adding 50 µL Reaction Mix to all wells. Carefully shake the plate for a few seconds to mix.
3. Seal the plate with a plate sealer or plastic wrap to reduce evaporation. Incubate the plate for 30 minutes at 37°C with mixing in the dark.
4. Measure the absorbance at570 nm using a plate reader.