Description
alpha-Ketoglutarate Dehydrogenase Complex (αKGDC) Activity Assay Kit
The Assay Genie alpha-KGDC Activity Assay Kit provides a simple colorimetric method for measuring alpha-ketoglutarate dehydrogenase complex (αKGDC, also known as 2-oxoglutarate dehydrogenase complex, OGDC/OGDH) activity in cell and tissue samples. αKGDC is a mitochondrial multi-enzyme complex that converts α-ketoglutarate to succinyl-CoA with production of NADH, and is a key rate-controlling step of the tricarboxylic acid (TCA) cycle.
Because αKGDC is associated with mitochondrial membranes, the kit includes both a Lysis Buffer and an Extraction Buffer to prepare an enriched membrane fraction. NADH generated by the complex reduces the tetrazolium dye INT to a red formazan, read at 492 nm in a standard 96-well plate, with a matched no-substrate control for every sample.
| Product Name: | alpha-KGDC Assay Kit |
| SKU: | BR00003 |
| Kit Size: | 100 wells |
| Detection Method: | Colorimetric (INT → formazan, OD 492 nm) |
| Sample Types: | Membrane fraction of cells and tissues (prepared with the included buffers) |
| Recommended Protein: | 0.2–2 mg/ml |
| Incubation Time: | 60 or 120 min at 37 °C |
| Units: | IU/L (µmol/L/min); normalise to protein or GAPDH |
| Equipment Required: | Microplate reader (492 nm) |
| Storage: | -80 °C (see component storage) |
| Intended Use: | For research use only (RUO). Not for diagnostic procedures. |
- Direct activity calculation in IU/L, no standard curve required
- Two-buffer workflow enriches the mitochondrial membrane fraction
- Lysate supernatant can be kept for hexokinase, GAPDH or LDH assays
- Per-sample no-substrate control removes background signal
- Simple 96-well, mix-and-read format
- αKGDC oxidatively decarboxylates α-ketoglutarate to succinyl-CoA, reducing NAD+ to NADH.
- NADH drives the reduction of the tetrazolium salt INT to a coloured formazan. Formazan has an absorbance maximum at 492 nm (ε = 18 mM-1cm-1), so activity is calculated directly in IU/L (µmol/L/min) without a standard curve.
- Activity is calculated from ΔOD (reaction − control): IU/L = ΔOD × 8.1 (60 min) or × 4.05 (120 min).
- Cells: ~106 cells lysed in ~0.1 ml 1x Lysis Buffer; centrifuge and resuspend the pellet in ~50 µl 1x Extraction Buffer.
- Tissue: ~5 mg tissue homogenised in ~0.5 ml 1x Lysis Buffer; resuspend the pellet in ~0.25 ml 1x Extraction Buffer.
- Centrifuge at ~5,000 rpm for 1 min and use the supernatant for the assay.
| Component | Size | Storage |
| αKGDC Assay Solution | 5 ml | -80 °C, protect from light |
| 20x αKGDC Substrate | 0.25 ml | -20 °C or -80 °C |
| 20x Cofactors | 0.25 ml | -20 °C or -80 °C, protect from light |
| 10x Lysis Buffer | 25 ml | 4 °C |
| 10x Extraction Buffer | 20 ml | 4 °C |
- TCA cycle flux and mitochondrial function studies
- Oxidative stress research (αKGDC is highly sensitive to oxidative inactivation)
- Neurodegeneration and brain energy metabolism research
- Cancer metabolism and α-ketoglutarate signalling
Why does the alpha-KGDC kit use an extraction buffer?
αKGDC is associated with the mitochondrial inner membrane. The Extraction Buffer solubilises the membrane fraction left after cytosolic lysis, enriching the complex for measurement.
Do I need a standard curve?
No. Activity is calculated directly from ΔOD at 492 nm using the formazan extinction coefficient, giving results in IU/L.
What equipment do I need?
A microplate reader at 492 nm, a 37 °C (non-CO2) incubator, a refrigerated microcentrifuge and a plain 96-well plate.
