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Pyruvate Colorimetric/Fluorometric Assay Kit

based on 32 citations in multiple journalsPyruvate Colorimetric/Fluorometric Assay Kit325 5
Simple Assay, HTS
Catalog #: K609

In stock

$475.00

Product Details

Cat # +Size K609-100
Size 100 assays
Detection Method Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm)
Species Reactivity Mammalian
Applications The kit detects 1 µM to 10 mM pyruvate.
Features & Benefits • Simple procedure; takes ~40 minutes
• Fast and convenient
• Kit contains all necessary reagents for accurate measurement of pyruvate concentrations in biological samples
Kit Components • Pyruvate Assay Buffer
• Pyruvate Probe (in DMSO)
• Pyruvate Enzyme Mix
• Pyruvate Standard (100 nmol/µl)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

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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What is the process to use this kit with adherent cells?
If cells are collected by Trypsinization,it is essential to neutralize the Trypsin with medium. Then all media must be removed and cells washed with PBS. For measuring pyruvate start with 1-2x106 cells, suspend the cell pellet in 3-4 volumes of the Pyruvate assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Load the supernatant unto a 10kda spin column for deproteinzation. Use the eluate for your subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.
The OD values are very low. What could be the problem?
The most common reason for low OD values is that the Assay buffer was cold, which led to a slow reaction.
Can an acid precipitation method be used for removing proteins before this assay?
Yes, A Perchloric acid method can be used. Please refer to our kit K808.
Is it possible that the pyruvate in the sample is degraded with time?
Yes, pyruvate is lost fairly rapidly and can get used up by cellular enzymes. It is essential to deproteinize the samples as soon as possible.
Should the samples be deproteinized and stored?
Yes, it is recommended that cell/tissue lysates or media is deproteinized and then stored at -80C if needed.
How much pyruvate is there is serum versus whole blood?
Typically, normal human serum has a pyruvate concentration in the range of 60-150 µM and normal human blood has a pyruvate concentration in the range of 35-100 µM.
What kind of interfering substances can affect the assay results? Undiluted deproteinized samples show values above the expected/reported range.
Compounds such as lactate and a-keto acids in the samples are known to have potential to interfere with the measurement of pyruvate, particularly, 2-Oxobutyrate and oxaloacetate. This could explain why the undiluted deproteinized samples suffer from overestimation.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
Can individual components of this kit be purchased separately?
Yes, any of the kit's components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Is it possible to use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
Can this kit be used to measure pyruvate in yeast or bacterial cells?
This assay protocol has been optimized for samples of mammalian origin but theoretically this assay should work with samples from multiple origins including bacteria and yeast. Special lysis reagents might be required and the sample amount might need optimization for best results.
Will the phenol red in the media affect the assay readout?
Very low amounts of media are used for each sample. This will generate a very low background at the best. Please use only media as a background control and subtract this reading from all sample readings to accommodate for the phenol red.
Why is it essential to use less probe for the fluorometric assay?
The fluorometric assay is at least 10 times more sensitive and hence too much probe can saturate the detector of the fluorometer. Therefore 5 times less probe is used.
Chem et al., Mitochondrial Citrate Transporter-dependent Metabolic Signature in the 22q11.2 Deletion Syndrome. J. Biol. Chem., Sep 2015; 290: 23240 - 23253.
Kim et al., Mice expressing reduced levels of hepatic glucose-6-phosphatase-α activity do not develop age-related insulin resistance or obesity.  Hum.  Mol. Genet., Sep 2015; 24: 5115 - 5125.
Chen et al., Cadmium toxicity induces ER stress and apoptosis via impairing energy homoeostasis in cardiomyocytes. Biosci. Rep., Jun 2015; 35: e00214.
Palamiuc et al., A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. EMBO Mol Med., May 2015; 7: 526 - 546.
Chettimada et al., Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension.Am J Physiol Lung Cell Mol Physiol, Feb 2015; 308: L287 - L300.
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