Isocitrate Dehydrogenase (IDH) is a metabolic enzyme that decarboxylates isocitrate into α-ketoglutarate producing CO₂ and generating NADH or NADPH. By producing these cofactors, IDH provides essential antioxidant molecules that defend cells against oxidative stress. The cytoplasmic and peroxisomal IDH1 isoform generates NADPH and when somatic mutations occur it drives glioma development, acute myeloid leukemia (AML), and other malignancies. Within the mitochondria, IDH2 isoform also produces NADPH and its mutations are linked to hematologic cancers, cholangiocarcinoma, and Ollier disease. IDH3 isoform functions within the mitochondrial Krebs cycle to generate NADH. Germline mutations in IDH3 triggers inherited retinal degeneration and macular pseudocoloboma. Monitoring IDH activity is critical in diagnostic for evaluating cellular health and managing cancers.

Isocitrate Dehydrogenase Activity Assay Kit provides a simple, reproducible, and sensitive tool for measuring NAD-dependent, NADPH-dependent or both IDHs activity in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay is based on a coupled enzyme reaction. Isocitrate is decarboxylated by IDH to produce NADH, NADPH, or both which reacts to form a colored product that can be quantified using a spectrophotometer or a microtiter plate reader at 450 nm. The change in absorbance is directly proportional to IDH activity in the sample.

• Microplate reader capable of measuring absorbance at 450 nm at 37°C
• 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
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the diluted NADH standards, samples, or positive control to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix (+SUB) to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix (-SUB) to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Kinetic reading: Immediately start measuring absorbance at 450 nm continuously at 37°C and record data every 2 minutes for 30 minutes.