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Amplite® Fluorimetric α-Ketoglutarate Quantitation Kit
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. Amplite®™ Fluorimetric α-Ketoglutarate Quantitation Kit offers a sensitive fluorimetric assay for quantifying α-ketoglutarate in biological samples. It utilizes an enzyme coupled reaction that releases hydrogen peroxide, which can be detected with Amplite®™ Red at Ex/Em ~540/590 nm (red fluorescence).
Example protocol
AT A GLANCE
Protocol Summary
- Prepare α-Ketoglutarate standards or test samples (50 µL)
- Add α-Ketoglutarate Assay working solution (50 µL)
- Incubate at 37 °C for 30 to 60 minutes
- Read fluorescence intensity at Ex/Em = 540/590 nm
PREPARATION OF STOCK SOLUTIONS
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.
Note Store unused Amplite™ Red stock solution (200X) at -20°C, avoid light and repeated freeze-thaw cycles.
Amplite™ Red stock solution (200X)
Add 50 µL of DMSO (Component E) into one vial of Amplite™ Red (Component A) and mix them well.Note Store unused Amplite™ Red stock solution (200X) at -20°C, avoid light and repeated freeze-thaw cycles.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/10087
https://www.aatbio.com/tools/serial-dilution/10087
alpha-Ketoglutarate standard
Add 2.5 uL of alpha-Ketoglutarate standard in 225 uL of PBS buffer to make 100 uM. Perform 1:3 serial dilutions to get approximately 33, 11, 3.7, 1.23, 0.41 and 0.1 μM serially diluted α-ketoglutarate standards.PREPARATION OF WORKING SOLUTION
α-Ketoglutarate Assay working solution
Add 5 mL Assay Buffer (Component C) into one Enzyme Mix 1 bottle (Component B1) and mix well. Add 100 μL of ddH2O into one Enzyme Mix 2 vial (Component B2) and mix well. Transfer entire vial (100 μL) of Enzyme Mix 2 and 25 µL of 200X Amplite™ Red stock solution (200X) into the vial of Enzyme Mix 1 and mix well.Note The 5 mL working solution is enough for one 96-wells plate. It is not stable, use it promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of α-Ketoglutarate standards and test samples in a solid black 96-well microplate. AKG= α-Ketoglutarate Standards (AKG1 - AKG7, 0.1 to 100 µM), BL=Blank Control, TS=Test Samples.
Table 2. Reagent composition for each well.
| BL | BL | TS | TS |
| AKG1 | AKG1 | ... | ... |
| AKG2 | AKG2 | ... | ... |
| AKG3 | AKG3 | ||
| AKG4 | AKG4 | ||
| AKG5 | AKG5 | ||
| AKG6 | AKG6 | ||
| AKG7 | AKG7 |
| Well | Volume | Reagent |
| AKG1 - AKG7 | 50 µL | Serial Dilutions (0.1 to 100 µM) |
| BL | 50 µL | Assay Buffer (Component C) |
| TS | 50 µL | test sample |
- 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.
- Add 50 µL of α-Ketoglutarate Assay working solution to each well of α-Ketoglutarate standard, blank control, and test samples to make the total 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.
- Incubate the reaction at 37 ºC for 30 - 60 minutes.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
Citations
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
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
References
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
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
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
Authors: Hangasky JA, Taabazuing CY, Martin CB, Eron SJ, Knapp MJ.
Journal: J Inorg Biochem (2017): 26
Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward alpha-Ketoglutarate
Authors: Martin-Mora D, Ortega A, Reyes-Darias JA, Garcia V, Lopez-Farfan D, Matilla MA, Krell T.
Journal: Front Microbiol (2016): 1937
Authors: Martin-Mora D, Ortega A, Reyes-Darias JA, Garcia V, Lopez-Farfan D, Matilla MA, Krell T.
Journal: Front Microbiol (2016): 1937
Dietary Supplementation with alpha-Ketoglutarate Activates mTOR Signaling and Enhances Energy Status in Skeletal Muscle of Lipopolysaccharide-Challenged Piglets
Authors: Wang L, Yi D, Hou Y, Ding B, Li K, Li B, Zhu H, Liu Y, Wu G.
Journal: J Nutr (2016): 1514
Authors: Wang L, Yi D, Hou Y, Ding B, Li K, Li B, Zhu H, Liu Y, Wu G.
Journal: J Nutr (2016): 1514
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