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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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Please review our privacy policy. Fluorescence excitation and emission were performed at nm and nm, respectively, for experiments requiring high sensitivity.

We have recently reported a new sensitive and selective fluorimetric assay for pyruvate measurement using Amplex Red as a fluorogenic dye. 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 H 2 O 2 in the presence of horseradish peroxidase HRP to brightly fluorescent resorufin [ 3 ].

For these reasons, the reactions should be performed at pH 7—8. Free Radic Biol Med. Springer-Verlag; Berlin; New York: The background intensity was subtracted using the value of the no pyruvate control for all sample readings.

As shown in Fig. Properties of the Amplex UltraRed assay.

amplrx Therefore, the enzymatic reactions are favorable in acidic buffer, while the fluorogenic reaction is more efficient in a neutral or weakly basic solution.


Regulation of store-operated calcium channels by the intermediary metabolite pyruvic acid. Group 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.

Error bars are not shown because their sizes are smaller than the symbols. Contact Us to ask amplfx question, provide feedback, or report a problem. Open in a separate window.

Support Center Support Hltra. However, the fluorescence of the reaction product, resorufin, is pH-dependent, which limits the sensitivity of the assay at pH 6. However, the absorption and fluorescence of resorufin are pH-dependent. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.

Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

A sensitive fluorimetric assay for pyruvate. Pyruvate protects neurons against hydrogen peroxide-induced toxicity. National Center for Biotechnology InformationU.

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The results show that Amplex UltraRed improves the performance of pyruvate assay, providing enhanced signal sensitivity. Therefore, we recommend fresh preparations of Amplex UltraRed aqueous solutions prior to assays.

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The side reaction between H 2 O ampkex and pyruvate can be effectively suppressed in acidic buffer pH 5. Concentrations of metabolites in animal tissues. An official website of the United States government. The specificity of the assay is shown in panel C. Louis, MO unless otherwise noted.

Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

As HClO 4 is commonly used as a deproteinizing reagent in pyruvate sample preparation, we tested its effect on this assay. For bacterial pyruvate oxidase, one unit will produce 1. The excitation wavelength is nm. Due to a lapse in appropriations, EPA websites will not be regularly fed. As a service to our customers we are providing this early version of the manuscript.

This is a PDF file of an unedited manuscript that has been accepted for publication. We also tested the stability of Amplex UltraRed. We next evaluated the selectivity of the method by performing the same assay with hltra PEPlactate, acetoacetate, ketoglutarate, 2-oxobutyrate, oxaloacetate as samples. The pH optimum of HRP is in the range of 6.