Alpha-ketoglutarate (α-KG) plays a critical role in cellular metabolism and energy production within the Krebs cycle, while serving as a vital precursor for amino acid synthesis. Altered α-ketoglutarate metabolism is linked to hyperinsulinism-hyperammonemia syndrome and nutrional deficiences like a shortage of B vitamins. Reduced tissue levels of α-ketoglutarate are a natural marker of aging, but can also indicate Alzheimer's, Parkinson’s, and diabetes due to altered glutamine metabolism or downregulation of upstream producing enzymes like malate-aspartate shuttle. Measuring α-ketoglutarate levels is important in understanding epigenetic regulation, mitochondrial metabolic flux, and age-related cellular decline.

Alpha-Ketoglutarate Assay Kit provides a simple, reproducible, and sensitive tool of measuring α-KG 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. Alpha-Ketoglutarate is catalyzed by an enzyme to produce a colored product which can be quantified using a spectrophotometer or a microtiter plate reader at 570 nm. The optical density measured at 570 nm is directly proportional to the concentration of α-ketoglutarate 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
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• Orbital shaker capable of 37°C
• MilliQ water or another type of double distilled water (ddH₂O)
• Perchloric acid (HClO₄) (PCA)
• Potassium hydroxide (KOH)

1. Add 50 µL of the diluted α-KG Standards or samples to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. 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.
5. Measure the absorbance at 570 nm using a plate reader.