Acetoacetate, a ketone body, is an important intermediate in ketogenesis, where it is produced from acetyl-CoA in the liver to serve as a precursor to other ketone bodies – beta-hydroxybutyrate and acetone. It is used as an alternative energy source during low glucose availability, such as low-carbohydrate diets and insulin-resistant conditions like diabetes. Monitoring acetoacetate levels is important for evaluating ketosis severity and ketoacidosis.

Acetoacetate Assay Kit provides a simple, reproducible, and sensitive tool of measuring acetoacetate concentration in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay employs a non-enzymatic reaction involving acetoacetate reacting with a substrate to produce a colored product that is measurable at 550 nm. The optical density measured at 550 nm is directly proportional to the concentration of acetoacetate present in the sample.

• Microplate reader capable of measuring absorbance at 550 nm at 25°C
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 110 µL of the diluted Acetoacetate Standards or samples to the 96-well microtiter plate.
2. Add 90 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 90 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Kinetic reading: Immediately begin reading absorbance at 550 nm continuously at 25°C and record data every 2 minutes for 20 minutes.