AAT Bioquest

Amplite® Colorimetric α-Ketoglutarate Quantitation Kit

Alpha-ketoglutarate dose response was measured with Amplite® Colorimetric α-Ketoglutarate Quantitation Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices) with path check on mode.
Alpha-ketoglutarate dose response was measured with Amplite® Colorimetric α-Ketoglutarate Quantitation Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices) with path check on mode.
Alpha-ketoglutarate dose response was measured with Amplite® Colorimetric α-Ketoglutarate Quantitation Kit in a 96-well clear bottom plate using a SpectraMax microplate reader (Molecular Devices) with path check on mode.
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Alpha-ketoglutarate (α-ketoglutarate) is a key molecule in the Krebs cycle determining the overall rate of the citric acid cycle of the organism. As a precursor of glutamate and glutamine, α-ketoglutarate is a central metabolic fuel for cells of the gastrointestinal tract as well. It can decrease protein catabolism and increase protein synthesis to enhance bone tissue formation in the skeletal muscles and can be used in clinical applications. Alpha-ketoglutarate is used for kidney disease; intestinal and stomach disorders, including bacterial infections; liver problems; cataracts; and recurring yeast infections. It is also used for improving the way kidney patients receiving hemodialysis treatments process protein. AAT Bioquest's Amplite® Colorimetric α-Ketoglutarate Quantitation Kit offers a sensitive colorimetric assay for quantifying α-ketoglutarate in biological samples. It utilizes an enzyme coupled reaction that releases hydrogen peroxide, which can be detected by Amplite® Red in an absorbance microplate reader at 570 nm.


Absorbance microplate reader

Absorbance575 nm
Recommended plateClear bottom
Instrument specification(s)Path check


Example protocol


Protocol summary

  1. Prepare test samples along with serially diluted α-ketoglutarate standards (50 µL)
  2. Add equal volume of α-Ketoglutarate working solution (50 µL)
  3. Incubate at 37°C for 60 - 90 minutes
  4. Monitor absorbance intensity at 570 nm

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (200X):
Add 50 µL of DMSO (Component E) into the vial of Amplite™ Red (Component A) to make 200X stock solution.


α-Ketoglutarate standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10085

Add 10 µL of 10 mM α-Ketoglutarate Standard (Component D) into 990 µL of PBS to get 100 µM α-ketoglutarate standard solution (AKG7). Then perform 1:2 serial dilutions to get serially diluted α-ketoglutarate standards (AKG6 - AKG1).


1. Add 5 mL Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1) and mix well.

2. Add 100 μL of ddH2O into one Enzyme Mix 2 vial (Component B2) and mix well.

3. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 µL of 200X Amplite™ Red stock solution into the vial of Enzyme Mix 1 and mix well to make α-Ketoglutarate working solution. Note: The 5 mL α-Ketoglutarate working solution is enough for one 96-well plate. It is not stable, use it promptly.


Table 1. Layout of α-ketoglutarate standards and test samples in a 96-well clear bottom microplate. AKG= α-Ketoglutarate Standard (AKG1 - AKG7, 1.563 to 100 µM), BL=Blank Control, TS=Test Sample. 


Table 2. Reagent composition for each well.

AKG1 - AKG750 µLSerial Dilutions (1.563 to 100 µM)
BL50 µLAssay Buffer (Component C)
TS50 µLtest sample
  1. Prepare α-ketoglutarate standards (AKG), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of α-Ketoglutarate working solution to each well of α-ketoglutarate standard, blank control, and test samples to make the total α-ketoglutarate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of α-Ketoglutarate working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction mixture at 37°C for 60 - 90 minutes.

  4. Monitor the absorbance increase with an absorbance plate reader with path check on at OD of 570 nm.



View all 1 citations: Citation Explorer
Alpha-ketoglutarate alleviates cadmium-induced inflammation by inhibiting the HIF1A-TNFAIP3 pathway in hepatocytes
Authors: Jia, Yinzhao and Yin, Chuanzheng and Ke, Wenbo and Liu, Jing and Guo, Bing and Wang, Xiaofei and Zhao, Peng and Hu, Shaobo and Zhang, Chen and Li, Xuan and others,
Journal: Science of The Total Environment (2023): 163069


View all 40 references: Citation Explorer
Loss of Nardilysin, a Mitochondrial Co-chaperone for alpha-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration
Authors: Yoon WH, S and oval H, Nagarkar-Jaiswal S, Jaiswal M, Yamamoto S, Haelterman NA, Putluri N, Putluri V, Sreekumar A, Tos T, Aksoy A, Donti T, Graham BH, Ohno M, Nishi E, Hunter J, Muzny DM, Carmichael J, Shen J, Arboleda VA, Nelson SF, Wangler MF, Karaca E, Lupski JR, Bellen HJ.
Journal: Neuron (2017): 115
Dietary alpha-ketoglutarate promotes higher protein and lower triacylglyceride levels and induces oxidative stress in larvae and young adults but not in middle-aged Drosophila melanogaster
Authors: Bayliak MM, Lylyk MP, Shmihel HV, Sorochynska OM, Semchyshyn OI, Storey JM, Storey KB, Lushchak VI.
Journal: Comp Biochem Physiol A Mol Integr Physiol (2017): 28
The facial triad in the alpha-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation
Authors: Hangasky JA, Taabazuing CY, Martin CB, Eron SJ, Knapp MJ.
Journal: J Inorg Biochem (2017): 26
Comparative genomics analysis of a series of Yarrowia lipolytica WSH-Z06 mutants with varied capacity for alpha-ketoglutarate production
Authors: Zeng W, Fang F, Liu S, Du G, Chen J, Zhou J.
Journal: J Biotechnol (2016): 76
Applying pathway engineering to enhance production of alpha-ketoglutarate in Yarrowia lipolytica
Authors: Guo H, Su S, Madzak C, Zhou J, Chen H, Chen G.
Journal: Appl Microbiol Biotechnol (2016): 9875
Electronic Structure of the Ferryl Intermediate in the alpha-Ketoglutarate Dependent Non-Heme Iron Halogenase SyrB2: Contributions to H Atom Abstraction Reactivity
Authors: Srnec M, Wong SD, Matthews ML, Krebs C, Bollinger JM, Jr., Solomon EI.
Journal: J Am Chem Soc (2016): 5110
Registered report: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate
Authors: Fiehn O, Showalter MR, Schaner-Tooley CE.
Journal: Elife (2016)
Reductions in the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental
Authors: Chen H, Denton TT, Xu H, Calingasan N, Beal MF, Gibson GE.
Journal: J Neurochem (2016): 823
Mild mitochondrial metabolic deficits by alpha-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease
Authors: Banerjee K, Munshi S, Xu H, Frank DE, Chen HL, Chu CT, Yang J, Cho S, Kagan VE, Denton TT, Tyurina YY, Jiang JF, Gibson GE.
Journal: Neurochem Int (2016): 32
Mechanism of O2 Activation by alpha-Ketoglutarate Dependent Oxygenases Revisited. A Quantum Chemical Study
Authors: Wojcik A, Radon M, Borowski T.
Journal: J Phys Chem A (2016): 1261