AMPLEX ULTRA RED PDF

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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Therefore, we recommend fresh preparations of Amplex UltraRed aqueous solutions prior to assays. We also tested the stability of Amplex UltraRed. Open in a separate window. Abstract Pyruvate, which is produced in the final step of glycolysis, participates in anabolic metabolism, catabolic metabolism, and signal transduction. 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 ].

Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

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. This is a PDF file of an unedited manuscript that has been accepted for rwd. Each point is the mean of triplicate measurements. The role of phosphometabolites in cell proliferation, energy metabolism, and tumor therapy. Therefore, the enzymatic reactions are favorable in acidic buffer, while the fluorogenic reaction is more efficient in a neutral or weakly basic solution.

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

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The amlpex of sodium pyruvate in assessing damage produced by hydrogen peroxide. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. We have recently reported a new sensitive and selective fluorimetric assay for pyruvate measurement using Amplex Reed as a fluorogenic dye.

Error bars are not shown because their sizes are smaller than the symbols. Lamprecht W, Heinz F. The publisher’s final edited version of this article is available at Anal Biochem.

Pyruvate is produced by pyruvate kinase EC2. The first column in Fig. The pH optimum of HRP is in the range of 6. National Center for Biotechnology InformationU. We next evaluated the selectivity of the method by performing the same assay with phosphoenolpyruvate PEPlactate, acetoacetate, ketoglutarate, 2-oxobutyrate, oxaloacetate as samples. The specificity of the assay is shown in panel C. Pyruvate protects neurons against hydrogen peroxide-induced toxicity. Louis, MO unless otherwise noted.

Support Center Support Center. Author information Copyright and License information Disclaimer. Free Radic Biol Med. However, its background fluorescence is higher. Methods of enzymatic analysis.

Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection

The NaClO 4 concentration is As a service to our customers we are providing this rde version of the manuscript. Properties of the Amplex UltraRed assay.

In addition, H 2 O 2 has been reported to cause a nonenzymatic and stoichiometric decarboxylation of pyruvate [ 56 ]. To overcome this problem, we examined this fluorimetric assay with the new dye Amplex UltraRed Cat. However, the fluorescence of the reaction product, resorufin, is pH-dependent, which limits the sensitivity of the assay at pH 6.

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Academic Press; New York: The results show that Amplex UltraRed improves the performance of pyruvate assay, providing enhanced signal sensitivity. 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. Please note that all information on the EPA website may not be up to date, and transactions and inquiries submitted hltra the EPA website may not be processed or responded to until appropriations are enacted.

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

Pyruvate, which is produced in amplec final step of glycolysis, participates in anabolic metabolism, catabolic metabolism, and signal transduction. The linearity of these data are excellent, as judged by the coefficients of determination of 0. Springer-Verlag; Berlin; New York: A sensitive fluorimetric assay for pyruvate.

Our results show that Amplex UltraRed improves the performance of the assay, providing brighter fluorescence and enhanced sensitivity. As HClO 4 is commonly used as a deproteinizing reagent in pyruvate sample preparation, we tested its effect on this assay. We’ve made some changes to EPA.

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