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Pyruvate Colorimetric/Fluorometric Assay Kit Kit

BCA Reactivity: Chemical Fluorometric, Colorimetric Adherent Cell Culture, Cell Culture Cells, Cell Samples, Saliva, Tissue Samples
Pubmed (26)
Catalog No. ABIN411703
$515.00
Plus shipping costs $45.00
100 tests
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  • Target
    Pyruvate
    Reactivity
    Chemical
    Detection Method
    Fluorometric, Colorimetric
    Detection Range
    0.001-10 mM
    Minimum Detection Limit
    0.001 mM
    Application
    Biochemical Assay (BCA)
    Sample Type
    Adherent Cell Culture, Cell Culture Cells, Cell Samples, Saliva, Tissue Samples
    Specificity
    The Kit provides a simple, direct and automation-ready procedure for measuring pyruvate concentration in various biological samples such as blood, cells, culture and fermentation media, etc. In the assay, pyruvate is oxidized by pyruvate oxidase via enzyme reactions to generate color (at lambda = 570 nm) and fluorescence (at Ex/Em = 535/587 nm). Since the color or fluorescence intensity is proportional to pyruvate content, the pyruvate concentration can be accurately measured. The kit detects 1-10000 ?M pyruvate.
    Characteristics
    Pyruvate Assay Kit: Colorimetric & Fluorometric Assay for measuring pyruvate concentration in various biological samples such as serum, saliva, tissues & adherent or suspension cells within 40 min. Convenient, Simple & Sensitive.
    Components
    Pyruvate Assay Buffer
    Pyruvate Probe (in DMSO)
    Pyruvate Enzyme Mix
    Pyruvate Standard (100 nmol/μl)
  • Application Notes
    The kit detects 1 μM to 10 mM pyruvate.
    Comment

    Further details regarding sample type:

    • Animal tissues
    • Cell culture: adherent or suspension cells
    • Serum, saliva

    Assay Time
    < 1 h
    Protocol
    1. Standard Curve Preparations:
    Colorimetric assay: Dilute the Pyruvate Standard to 1 nM/µL by adding 10 µL of the Standard to 990 µL of Pyruvate Assay Buffer, mix well.
    Fluorometric assay: Dilute the Pyruvate Standard to 1 nM/µL as for the colorimetric assay. Then dilute the standard another 10-fold to 0.1 nM/µL by taking 10 µL into 90 µL of Pyruvate Assay Buffer.
    Mix well. Add 0, 2, 4, 6, 8, 10 µL into a series of standards wells. Adjust volume to 50 µL/well with Pyruvate Assay Buffer to generate 0, 2, 4, 6, 8, 10 nM/well of the Pyruvate Standard for the colorimetric assay (0, 0.2, 0.4, 0.6, 0.8, 1.0 nM/well for the fluorometric assay).
    2. Sample Preparations: Prepare test samples in 50 µL/well with Pyruvate Assay Buffer in a 96- well plate. Serum can be directly added into sample wells, and adjust volume to 50 µL/well with Pyruvate Assay Buffer (serum contains approx. 50-100 pM/µL pyruvate). Tissues or cells can be extracted with 4 volume of the Pyruvate Assay Buffer, centrifuge to get clear pyruvate extract. We suggest using several doses of your sample to ensure the readings are within the standard curve range. Due to the presence of LDH in serum, care must be taken during sample processing to prevent the conversion of pyruvate to lactate. Samples can be deproteinized by 10 kDa cutoff spin filter to remove proteins.
    3. Reaction Mix Preparation: Mix enough reagents for the number of assays performed. For each well, prepare a total 50 µL Reaction Mix containing the following components. Mix well before use: 46 µL Pyruvate Assay Buffer 2 µL Pyruvate Probe 2 µL Enzyme Mix
    4. Add 50 µL of the Reaction Mix to each well containing the Pyruvate Standard or test samples, mix well.
    5. Incubate the reaction for 30 minutes at room temperature, protect from light.
    6. Measure O.D. 570 nm for colorimetric assay or fluorescence at Ex/Em = 535/590 nm in a microplate reader.
    Calculation of Results

    Correct background by subtracting the value derived from the 0 pyruvate control from all sample readings (Note: The background reading can be significant and must be subtracted from sample readings).
    Plot standard curve nM/well vs. O.D. 570 nm readings. Then apply the sample readings to the standard curve to get pyruvate amount in the sample wells (Py).
    The pyruvate concentrations in the test samples:
    C = Py/Sv (nM/µL or mM)
    Where: Py is the amount of pyruvate (nmol) of your sample from standard curve. Sv is the sample volume (in µL) added into the sample well. Pyruvate molecular weight: 88.08. Pyruvate concentration in your sample can be expressed as nM/mL, or mg/mL, or mg/dL or mM (mM/liter). 1 mM = 8.81 mg/dL.

    Restrictions
    For Research Use only
  • Storage
    -20 °C
    Expiry Date
    12 months
  • Chettimada, Gupte, Rawat, Gebb, McMurtry, Gupte: "Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension." in: American journal of physiology. Lung cellular and molecular physiology, Vol. 308, Issue 3, pp. L287-300, 2015 (PubMed).

    Palamiuc, Schlagowski, Ngo, Vernay, Dirrig-Grosch, Henriques, Boutillier, Zoll, Echaniz-Laguna, Loeffler, René: "A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis." in: EMBO molecular medicine, Vol. 7, Issue 5, pp. 526-46, 2015 (PubMed).

    Negi, Bingham, Li, Borthakur: "A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation." in: Plant physiology, Vol. 164, Issue 2, pp. 922-34, 2014 (PubMed).

    Park, Mukherjee, Ito, Chaumeil, Jalbert, Gaensler, Ronen, Nelson, Pieper: "Changes in pyruvate metabolism detected by magnetic resonance imaging are linked to DNA damage and serve as a sensor of temozolomide response in glioblastoma cells." in: Cancer research, Vol. 74, Issue 23, pp. 7115-24, 2014 (PubMed).

    Ramière, Rodriguez, Enache, Lotteau, André, Diaz: "Activity of hexokinase is increased by its interaction with hepatitis C virus protein NS5A." in: Journal of virology, Vol. 88, Issue 6, pp. 3246-54, 2014 (PubMed).

    Zager, Johnson, Becker: "Renal cortical pyruvate depletion during AKI." in: Journal of the American Society of Nephrology : JASN, Vol. 25, Issue 5, pp. 998-1012, 2014 (PubMed).

    Fried, Behr, Jung: "Identification of a target gene and activating stimulus for the YpdA/YpdB histidine kinase/response regulator system in Escherichia coli." in: Journal of bacteriology, Vol. 195, Issue 4, pp. 807-15, 2013 (PubMed).

    Müller, MacEachran, Burd, Sathitsuksanoh, Bi, Yeh, Lee, Hillson, Chhabra, Singer, Beller: "Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones." in: Applied and environmental microbiology, Vol. 79, Issue 14, pp. 4433-9, 2013 (PubMed).

    Everitt, Hu, Ajmo, Rogers, Liang, Zhang, Yin, Choi, Bennett, You: "Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice." in: American journal of physiology. Gastrointestinal and liver physiology, Vol. 304, Issue 1, pp. G38-47, 2013 (PubMed).

    Yi, Xu, Hiller, Kim, Nickeleit, James, Maeda: "Reduced expression of lipoic acid synthase accelerates diabetic nephropathy." in: Journal of the American Society of Nephrology : JASN, Vol. 23, Issue 1, pp. 103-11, 2012 (PubMed).

    Gatza, Wahl, Opipari, Sundberg, Reddy, Liu, Glick, Ferrara: "Manipulating the bioenergetics of alloreactive T cells causes their selective apoptosis and arrests graft-versus-host disease." in: Science translational medicine, Vol. 3, Issue 67, pp. 67ra8, 2011 (PubMed).

    Huang, Jones, Luo, Sanderson, Soto, Abel, Cooksey, McClain: "Iron overload and diabetes risk: a shift from glucose to Fatty Acid oxidation and increased hepatic glucose production in a mouse model of hereditary hemochromatosis." in: Diabetes, Vol. 60, Issue 1, pp. 80-7, 2011 (PubMed).

    Pircher, Straganz, Ehehalt, Morrow, Tanguay, Jansen-Dürr: "Identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase." in: The Journal of biological chemistry, Vol. 286, Issue 42, pp. 36500-8, 2011 (PubMed).

    Zhang, Ren, Li, Ma, Wang: "A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay." in: Biological procedures online, Vol. 11, pp. 170-83, 2011 (PubMed).

    Odet, Gabel, Williams, London, Goldberg, Eddy: "Lactate dehydrogenase C and energy metabolism in mouse sperm." in: Biology of reproduction, Vol. 85, Issue 3, pp. 556-64, 2011 (PubMed).

    Modrego, Mounux, Mateos-Cuaceres, Martuinez-Luopez, Segura, Zamorano-Leuon, Sierra, Serrano, Macaya, Luopez-Farrue: "Effects of platelets on the protein expression in aortic segments. A proteomic approach." in: Journal of cellular biochemistry, 2010 (PubMed).

    Dateki, Kunitomo, Yoshioka, Yanai, Nakasono, Negishi: "Adaptive gene regulation of pyruvate dehydrogenase kinase isoenzyme 4 in hepatotoxic chemical-induced liver injury and its stimulatory potential for DNA repair and cell proliferation." in: Journal of receptor and signal transduction research, Vol. 31, Issue 1, pp. 85-95, 2010 (PubMed).

    Cho, Park, Keam, Choi, Cho, Hyun, Park, Lee: "DDS, 4,4'-diaminodiphenylsulfone, extends organismic lifespan." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, Issue 45, pp. 19326-31, 2010 (PubMed).

    Katz, Cohen-Or, Gophna, Ron: "The ubiquitous conserved glycopeptidase Gcp prevents accumulation of toxic glycated proteins." in: mBio, Vol. 1, Issue 3, 2010 (PubMed).

    Tan, Sourris, Harcourt, Thallas-Bonke, Penfold, Andrikopoulos, Thomas, OBrien, Bierhaus, Cooper, Forbes, Coughlan: "Disparate effects on renal and oxidative parameters following RAGE deletion, AGE accumulation inhibition, or dietary AGE control in experimental diabetic nephropathy." in: American journal of physiology. Renal physiology, Vol. 298, Issue 3, pp. F763-70, 2010 (PubMed).

  • Target
    Pyruvate
    Target Type
    Chemical
    Background
    Pyruvate is a central molecule in metabolism through which sugars enter the citric acid cycle. Pyruvate can be converted to carbohydrates during gluconeogenesis or to fatty acids via acetyl CoA. High levels of pyruvate are associated with liver disease and genetic disorders. Pyruvate has also been used to stimulate metabolism leading to loss of body weight. BioVision provides a simple, direct and automation-ready procedure for measuring pyruvate concentration in various biological samples such as blood, cells, culture and fermentation media, etc. In the assay, pyruvate is oxidized by pyruvate oxidase via enzyme reactions to generate color (λ= 570 nm) and fluorescence (at Ex/Em = 535/587 nm). Since the color or fluorescence intensity is proportional to pyruvate content, the pyruvate concentration can be accurately measured. The kit detects 1 μM to 10 mM pyruvate.
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