Pyruvate, which is produced in the final step of glycolysis, participates in anabolic metabolism, catabolic metabolism, and signal transduction. Invitrogen Molecular Probes Amplex UltraRed Reagent 5 x 1mg Electrophoresis, Western Blotting and ELISA:Western Blotting Reagents, Membranes. Invitrogen Molecular Probes Amplex Red/UltraRed Stop Reagent 1 set Life Sciences:Cell Analysis:Cell Based Assays:Cell Growth and Differentiation Reagents.

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We also tested the stability of Amplex UltraRed. National Center for Biotechnology InformationU. The document below is a presentation from one of the monthly meetings of EPA’s Computational Toxicology Communities of Practice, a group composed of hundreds of people from over 50 public and private sector organizations. An excitation wavelength of nm is used. Academic Press; New York: The linearity of these data are excellent, as judged by the coefficients of determination of 0.

Pyruvate protects neurons against hydrogen peroxide-induced toxicity. Open in a separate window.

The specificity of the assay is shown in panel C. However, its background fluorescence is higher.

Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

All chemicals were purchased from Sigma-Aldrich St. However, the fluorescence of the reaction product, resorufin, is pH-dependent, which limits the sensitivity of the assay at pH 6.

Abstract Pyruvate, which is produced in the final step of glycolysis, participates in anabolic metabolism, catabolic metabolism, and signal transduction. As a service to our customers we are providing this early version of the manuscript. In addition, H 2 O 2 has been reported to cause a nonenzymatic and stoichiometric decarboxylation of pyruvate [ 56 ]. Concentrations of metabolites in utlra tissues.


The excitation wavelength is nm. We’ve made ulhra changes to EPA. As pyruvate production is associated with cell growth control, energy production, and intracellular signaling [ 12 ], it is important to have sensitive assays to evaluate pyruvate concentrations. A simple statistical parameter for use in evaluation and validation of high throughput screening assays.

Fluorescence excitation and emission were performed at nm and nm, respectively, for experiments requiring high sensitivity.

Analysis of High-throughput screening data from Amplex UltraRed (AUR) Thyroperoxidase (TPO) assays

However, the absorption and fluorescence of resorufin are pH-dependent. Jump to main content. For these reasons, the reactions should be performed at pH 7—8. The NaClO 4 concentration is A sensitive fluorimetric assay for pyruvate. Properties of the Amplex UltraRed assay.

The results show that Amplex UltraRed improves the performance of pyruvate assay, providing enhanced signal sensitivity. Ree members share an interest in encouraging computational toxicology usage and exposure science in helping to implement EPA’s mission of protecting human health and the environment. Due to a lapse in appropriations, EPA websites will not be regularly updated. Therefore, we recommend fresh preparations of Amplex UltraRed aqueous solutions prior to assays. Springer-Verlag; Berlin; New York: Methods of enzymatic analysis.

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.


Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

We next evaluated the selectivity of the method ultr performing the same assay with phosphoenolpyruvate PEPlactate, acetoacetate, ketoglutarate, 2-oxobutyrate, oxaloacetate as samples. Therefore, the enzymatic reactions are favorable in acidic buffer, while the fluorogenic reaction is more efficient in a neutral or ulrra basic solution. Recently, we reported a sensitive enzymatic fluorimetric assay for the determination of intracellular pyruvate based on the stoichiometric oxidization of non-fluorescent Amplex Red acetyl-3, 7-dihydroxyphenoxazine by Ampex 2 O 2 in the presence of horseradish peroxidase HRP to brightly fluorescent resorufin [ 3 ].

As HClO 4 is commonly used as a deproteinizing reagent in pyruvate sample preparation, we tested its effect on this assay. Author manuscript; available in PMC Aug 1. The background intensity was subtracted using the value of the no pyruvate control for all sample readings.

As shown in Fig. The first column in Fig. This is a PDF file of an unedited manuscript that has been accepted for publication.