Alpha-Ketoglutarate Colorimetric/Fluorometric Assay Kit

Details for Product No. ABIN411786, Supplier: Log in to see
Antigen
Reactivity
Mammalian
1
Detection Range
0.01-10 nM
Minimum Detection Limit
0.01 nM
Application
Biochemical Assay (BCA)
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Purpose α-Ketoglutarate Assay Kit provides a simple, sensitive and rapid means for quantifying α-KG in a variety of samples. In the assay, α-ketoglutarate is transaminated with the generation of pyruvate which is utilized to convert a nearly colorless probe to both color (λmax = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The α-Ketoglutarate Assay Kit is useful for detecting α-ketoglutarate in the range of 0.01 to 10 nmoles.
Sample Type Cell and Tissue Culture Supernatant, Plasma, Serum, Tissue Samples, Biological Fluids
Detection Method Fluorometric, Colorimetric
Specificity Alpha-Ketoglutarate Assay Kit provides a simple, sensitive and rapid means for quantifying alpha-KG in a variety of samples. In the assay, alpha-ketoglutarate is transaminated with the generation of pyruvate which is utilized to convert a nearly colorless probe to both color (lambda max = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The alpha-Ketoglutarate Assay Kit is useful for detecting alpha-ketoglutarate in the range of 0.01 to 10 nmoles.
Characteristics α-Ketoglutarate (α-KG) Assay Kit: Colorimetric & Fluorometric Assay for Quantifying α-KG in a variety of Samples within 40 min. Rapid, Convenient & Reliable.
Components α-KG Assay Buffer
α-KG OxiRed Probe (in DMSO)
α-KG Converting Enzyme
α-KG Development Enzyme Mix
α-KG Standard (10 μmol)
Background α-Ketoglutarate (α-KG) is a key intermediate in the Krebs cycle, coming after isocitrate and before succinyl CoA. Anaplerotic reactions replenish the cycle by synthesizing α-KG from transamination of glutamate, or through the action of glutamate dehydrogenase. α-KG is an important nitrogen transporter. Being a key intermediate, it is one of the organic acids measured in newborns as an indicator of inborn errors of metabolism.
Application Notes The α-Ketoglutarate Assay Kit is useful for detecting α-ketoglutarate in the range of 0.01 to 10 nmoles.
Comment

Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids

Assay Time < 1 h
Protocol 1. Standard Curve Preparations: Dilute the alpha-KG Standard to 1 nM/µL by adding 10 µL of the Standard to 990 µL of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 µL into a series of standards wells on a 96 well plate. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nM/well of the Standard.
2. Sample Preparation: Tissue (20 mg) or cells (2x10^6 ) are rapidly homogenized with 100 µL of ice cold PBS or other buffer (pH 6.5-8). Enzymes in samples may interfere with the assay. We suggest deproteinizing samples using a perchloric acid/KOH protocol or 10 kD molecular weight cut off spin columns. Add 1-50 µL samples into duplicate wells of a 96-well plate and bring volume to 50 µL with Assay Buffer. We suggest testing several doses of your samples to ensure readings are within the linear range.
3. Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µL Reaction Mix containing: Sample Bkgd. Control* alpha-KG Assay Buffer 44 µL 46 µL alpha-KG Converting Enzyme 2 µL ----- alpha-KG Enzyme Mix 2 µL 2 µL alpha-KG probe 2 µL 2 µL Add 50 µL of the Reaction Mix to each well containing the alpha-KG Standard, samples or background control*. * Note: Pyruvate generates background. If pyruvate is suspected in your sample, you can do the background control omitting the converting enzyme. The background control can be subtracted from the alpha-KG reading.
4. Incubate for 30 minutes at 37 °C, protect from light.
5. Measure OD at 570 nm or fluorescence using Ex/Em=535/587 nm.
Calculation of Results

Calculation: Correct background by subtracting the value of the 0 alpha-KG blank from all readings. Plot the standard curve. Apply the corrected sample readings to the standard curve to get alpha-KG amount in the sample wells. The alpha-KG concentrations in the test samples: C = Ay/Sv (nM/µL, or μM/mL, or mM) Where: Ay is the amount of alpha-KG (nmol) in your sample from the standard curve. Sv is the sample volume (µL) added to the sample well. alpha-Ketoglutarate molecular weight: 146.11 alpha-Ketoglutarate standard curve generated using this kit protocol.

Restrictions For Research Use only
Storage -20 °C
Expiry Date 12 months
Product cited in: Shao, Ahmad, Hodges, Kuang, Ratliff, Liu: "Inhibition of polo-like kinase 1 (Plk1) enhances the antineoplastic activity of metformin in prostate cancer." in: The Journal of biological chemistry, Vol. 290, Issue 4, pp. 2024-33, 2015 (PubMed).

Luo, Yoneda, Ohmori, Sasaki, Shimbo, Eto, Kato, Miyano, Kobayashi, Sasahira, Chihara, Kuniyasu: "Cancer usurps skeletal muscle as an energy repository." in: Cancer research, Vol. 74, Issue 1, pp. 330-40, 2014 (PubMed).

Jin, Reitman, Duncan, Spasojevic, Gooden, Rasheed, Yang, Lopez, He, McLendon, Bigner, Yan: "Disruption of wild-type IDH1 suppresses D-2-hydroxyglutarate production in IDH1-mutated gliomas." in: Cancer research, Vol. 73, Issue 2, pp. 496-501, 2013 (PubMed).

Sutendra, Dromparis, Wright, Bonnet, Haromy, Hao, McMurtry, Michalak, Vance, Sessa, Michelakis: "The role of Nogo and the mitochondria-endoplasmic reticulum unit in pulmonary hypertension." in: Science translational medicine, Vol. 3, Issue 88, pp. 88ra55, 2011 (PubMed).

Hu, Zhang, Wu, Sun, Levine, Feng: "Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, Issue 16, pp. 7455-60, 2010 (PubMed).