Reference: Hassaninasab A, et al. (2017) Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay. Anal Biochem 526:69-70

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Abstract


The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.

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Journal Article
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Hassaninasab A, Han GS, Carman GM
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