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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The first column in Fig. Therefore, we recommend fresh preparations of Amplex UltraRed aqueous solutions prior to assays. The side reaction between H 2 O 2 and amolex can be effectively suppressed in acidic buffer pH 5. The publisher’s final edited version of this article is available at Anal Biochem.

An official website of the United States government. Calibration curves for Amplex UltraRed using excitation wavelengths of nm A and nm B are shown. A sensitive fluorimetric assay for pyruvate. Pyruvate, which is produced in the final step of glycolysis, participates in ulra metabolism, catabolic metabolism, and signal transduction.

The background intensity was subtracted using the value of the no pyruvate control for all sample readings. In addition, H 2 O 2 has been reported to cause a nonenzymatic and stoichiometric decarboxylation of pyruvate [ 56 ]. Springer-Verlag; Berlin; New York: Error bars are not shown because their sizes are smaller than the symbols. Abstract Pyruvate, which is produced in the final step of glycolysis, participates in anabolic metabolism, catabolic metabolism, and signal transduction.


Methods of enzymatic analysis. The pH optimum of HRP is in the range of 6.

Louis, MO unless otherwise noted. The final reaction solution contained mM potassium phosphate with 1. Pyruvate is produced by pyruvate kinase EC2. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. National Center for Biotechnology InformationU.

Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

Our results show that Amplex UltraRed improves the performance of the assay, providing brighter fluorescence and enhanced sensitivity. 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 Rd 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.

In this case, the measured intensity is reduced by changing the excitation wavelength to nm where the linearity of the standard curve is improved in comparison to nm Fig. Author information Copyright and Zmplex information Disclaimer.

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

The importance of sodium pyruvate in assessing damage produced by hydrogen rex. 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. For these reasons, the reactions should be performed at pH 7—8. Author manuscript; available in PMC Aug 1.


Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

Contact Us to ask a question, provide feedback, or report a problem. Concentrations of metabolites in animal tissues.

As a service to our customers we are providing this early version of the manuscript. Pyruvate protects neurons against hydrogen peroxide-induced toxicity. The excitation wavelength is nm. The NaClO 4 concentration is 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. 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.

Due to a lapse in appropriations, EPA websites will not be regularly updated.

Therefore, the enzymatic reactions are favorable in acidic buffer, while the fluorogenic reaction is more efficient in a neutral or weakly basic solution. Each point is the mean of triplicate measurements. We also tested the stability of Amplex UltraRed. Regulation of store-operated calcium channels by the intermediary metabolite ultrx acid.

This is a PDF file of an unedited manuscript that has been accepted for publication.