Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay

Azam Hassaninasab, Gil Soo Han, George M. Carman

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)69-70
Number of pages2
JournalAnalytical Biochemistry
Volume526
DOIs
StatePublished - Jun 1 2017

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Keywords

  • Coupled enzyme assay
  • Fluorometric assay
  • Phosphatidic acid
  • Phosphatidic acid phosphatase
  • Yeast

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