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Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae
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Fluorescent Assays for Ceramide Synthase Activity.

Timothy A Couttas1, Anthony S Don2,3

  • 1Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia.

Methods in Molecular Biology (Clifton, N.J.)
|November 11, 2015
PubMed
Summary
This summary is machine-generated.

This study introduces a novel fluorescent assay for measuring ceramide synthase (CERS) activity, offering a safer alternative to radioactive methods. The assay

Keywords:
AssayCeramideCeramide synthaseFluorescent

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Lipid Metabolism

Background:

  • Ceramides are key sphingolipids influencing cell functions and implicated in diseases like diabetes and neurodegeneration.
  • Mammalian ceramide synthesis involves six ceramide synthases (CERS1-6) with distinct fatty acid specificities.
  • Traditional CERS activity assays rely on radioactive substrates, posing safety and handling concerns.

Purpose of the Study:

  • To describe a novel fluorescent assay for quantifying ceramide synthase (CERS) activity.
  • To provide methods for assay result quantification using Thin-Layer Chromatography (TLC) or High-Performance Liquid Chromatography (HPLC).

Main Methods:

  • Development of a fluorescent substrate-based assay for CERS activity.
  • Quantification of assay products using Thin-Layer Chromatography (TLC).
  • Quantification of assay products using High-Performance Liquid Chromatography (HPLC).

Main Results:

  • The fluorescent assay provides a viable alternative to radioactive methods for CERS activity measurement.
  • Established protocols for quantifying CERS activity using both TLC and HPLC are detailed.
  • The assay facilitates detailed analysis of CERS enzyme kinetics and substrate specificity.

Conclusions:

  • The described fluorescent CERS assay offers a safer and effective method for studying ceramide metabolism.
  • This assay and quantification techniques support research into the physiological and pathological roles of ceramides.
  • Advancements in ceramide research are facilitated by improved methodologies for CERS activity assessment.