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Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles

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Journal of Chemical Biology

Abstract

A wide range of methods exist for the on-plate detection of lipids resolved by thin layer chromatography. Fluorescence generally offers improvements in sensitivity over methods that use colorimetric or simple densitometric detection. In this paper, we report that a classic cupric sulfate charring protocol produces a fluorescent signal that sensitively and quantitatively detects a wide range of phospholipids, neutral lipids, and sterols after automated, multi-development high performance thin layer chromatography. The measured lower limits of detection and quantification, respectively, were, on average, 80 and 210 pmol for phospholipids and 43 fmol and 8.7 pmol for sterols. The simple, inexpensive, and highly sensitive approach described here was used to quantitatively analyze the lipid and sterol composition of sea urchin cortical vesicles, a stage-specific model system used to study the mechanism of regulated membrane fusion.

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Abbreviations

CV:

cortical vesicles

DAPC:

diacylphosphatidylcholine

MAG:

monoacylglycerol

DAG:

diacylglycerol

TAG:

triacylglycerol

DAPE:

diacylphosphatidylethanolamine

DAPS:

diacylphosphatidylserine

DAPI:

diacylphosphatidylinositol

DAPA:

diacylphosphatidic acid

DAPG:

diacylphosphatidylglycerol

FA:

fatty acids

CE:

cholesterol esters

SM:

sphingomyelin

CER:

ceramides

CA:

cardiolipin

HPTLC:

High-performance thin layer chromatography

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Acknowledgments

The authors wish to thank Paul P.M. Schnetkamp and Haider F. Altimimi for assistance with spectrofluorometry and Andrew Tang and Lauren Harris for helpful discussions. JRC acknowledges support from NSERC, CIHR, and AHFMR. MAC and DMB acknowledge support from NSERC.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada, T2N 4N1

    Matthew A. Churchward, David M. Brandman, Tatiana Rogasevskaia & Jens R. Coorssen

  2. Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada, T2N 4N1

    Jens R. Coorssen

  3. Department of Cell Biology and Anatomy, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada, T2N 4N1

    Jens R. Coorssen

  4. Hotchkiss Brain Institute, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada, T2N 4N1

    Jens R. Coorssen

  5. University of Western Sydney, School of Medicine (Bldg 30), Campbelltown, NSW, 1797, Australia

    Jens R. Coorssen

Authors
  1. Matthew A. Churchward
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  2. David M. Brandman
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  3. Tatiana Rogasevskaia
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  4. Jens R. Coorssen
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Corresponding author

Correspondence to Jens R. Coorssen.

Additional information

Matthew A. Churchward and David M. Brandman contributed equally to the study.

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Churchward, M.A., Brandman, D.M., Rogasevskaia, T. et al. Copper (II) sulfate charring for high sensitivity on-plate fluorescent detection of lipids and sterols: quantitative analyses of the composition of functional secretory vesicles. J Chem Biol 1, 79–87 (2008). https://doi.org/10.1007/s12154-008-0007-1

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  • DOI: https://doi.org/10.1007/s12154-008-0007-1

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