Total amounts of NAD+ and NADH were measured using a fluorescence-based cycling assay. The assay mixture consisted of 100 mM HEPES–NaOH (pH 8), 0.5 mM phenazine methosulfate, 0.4 mM resazurin, 10 mM of absolute ethanol and 5 U/mL alcohol dehydrogenase from yeast (Oriental Yeast, Japan). Appropriately diluted samples (10–40 µL) were mixed with assay mixture, and changes in fluorescence intensity over time were monitored with a microtiter plate and plate reader, GENios, (Tecan, Switzerland) at 37 °C (excitation wavelength, 540 nm; emission wavelength, 586 nm). Total concentration of NAD+ and NADH was quantified in comparison to a standard with known concentrations.
Alcohol dehydrogenase from yeast
Manufactured by Oriental Yeast
Sourced in Japan
Alcohol dehydrogenase from yeast is an enzyme that catalyzes the interconversion of alcohols and aldehydes or ketones. It is a commonly used laboratory reagent for various biochemical analyses and experiments.
Automatically generated - may contain errors
2 protocols using alcohol dehydrogenase from yeast
1
Fluorescence-based NAD+/NADH Quantification
2
Antioxidant Enzyme Assay Protocol
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4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) was from Sigma-Aldrich Japan (Tokyo, Japan). Xanthine oxidase, glutathione reductase (GR), NAD+, NADH, alcohol dehydrogenase from yeast and malate dehydrogenase from yeast were from Oriental Yeast Co., Ltd (Tokyo, Japan). The following were from Wako Pure Chemical Industries (Osaka, Japan): ferricytochrome c (from horse heart), hypoxanthine, cholesterol assay kit (Cholesterol E test), triglyceride assay kit (Triglyceride E test), Tiron, ascorbic acid, and glutathione. Dihydroethidium (DHE) was purchased from Life Technologies Japan Ltd. (Tokyo, Japan).
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