Amplite® Fluorimetric α-Ketoglutarate Quantitation Kit

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Telephone	1-800-990-8053
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

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).

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Solid black

Components

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

Important Thaw all the kit components to room temperature before starting the experiment.

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.

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

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.

BL	BL	TS	TS
AKG1	AKG1	...	...
AKG2	AKG2	...	...
AKG3	AKG3
AKG4	AKG4
AKG5	AKG5
AKG6	AKG6
AKG7	AKG7

Table 2. Reagent composition for each well.

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).

Images

Figure 1. Alpha-ketoglutarate dose response was measured with the Amplite® Fluorimetric α-Ketoglutarate Quantitation Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices) at Ex/Em = 540/590 nm, cutoff = 570 nm. As low as 1 µM of α-ketoglutarate can be detected with 30 minutes incubation.

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

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

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