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Goicoechea L, Arenas F, Castro F, et al. GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver. STAR Protoc. 2021;3(1):101068doi: 10.1016/j.xpro.2021.101068.
Goicoechea, L., Arenas, F., Castro, F., Nuñez, S., Torres, S., Garcia-Ruiz, C., & Fernandez-Checa, J. C. (2022). GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver. STAR protocols, 3(1), 101068. https://doi.org/10.1016/j.xpro.2021.101068
Goicoechea, Leire, et al. "GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver." STAR protocols vol. 3,1 (2022): 101068. doi: https://doi.org/10.1016/j.xpro.2021.101068
Goicoechea L, Arenas F, Castro F, Nuñez S, Torres S, Garcia-Ruiz C, Fernandez-Checa JC. GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver. STAR Protoc. 2021 Dec 29;3(1):101068. doi: 10.1016/j.xpro.2021.101068. eCollection 2022 Mar 18. PMID: 35024626; PMCID: PMC8728529.
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STAR Protoc. 2021 Dec 29;3(1):101068. doi: 10.1016/j.xpro.2021.101068. eCollection 2022 Mar 18.

GST-Perfringolysin O production for the localization and quantification of membrane cholesterol in human and mouse brain and liver.

STAR protocols

Leire Goicoechea, Fabian Arenas, Fernanda Castro, Susana Nuñez, Sandra Torres, Carmen Garcia-Ruiz, José C Fernandez-Checa

Affiliations

  1. Department of Cell Death and Proliferation, Institute of Biomedical Research of Barcelona (IIBB), CSIC, Barcelona, Spain.
  2. Liver Unit, Hospital Clinic i Provincial de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.
  3. Centro de Investigación Biomédica en Red (CIBEREHD), Barcelona, Spain.
  4. Research Center for ALPD, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

PMID: 35024626 PMCID: PMC8728529 DOI: 10.1016/j.xpro.2021.101068

Abstract

Abnormal cholesterol metabolism is linked to many neurodegenerative disorders. Here, we present a protocol for the production of a recombinant protein consisting of a Glutathione-S-Transferase tag fused with the Perfringolysin O (PFO). The GST-PFO tag enables analysis of the localization of cholesterol in subcellular membranes of human and mice brain and liver tissues. We have used this approach for samples from Niemann-Pick type C disease and non-alcoholic steatohepatitis models. The construct may also have applications for the diagnosis of cholesterol-accumulating disorders. For complete details on the use and execution of this protocol, please refer to Kwiatkowska et al. (2014).

© 2021 The Author(s).

Keywords: Biotechnology and bioengineering; Cell Biology; Cell Membrane; Health Sciences; Metabolism; Microscopy; Model Organisms; Molecular Biology; Molecular/Chemical Probes; Neuroscience; Protein expression and purification

Conflict of interest statement

The authors declare no competing interests.

References

  1. Aging (Albany NY). 2020 Jan 5;12(1):571-592 - PubMed
  2. Lancet. 2018 Mar 31;391(10127):1301-1314 - PubMed
  3. Biochim Biophys Acta. 1997 Jul 25;1327(2):222-30 - PubMed
  4. Methods Cell Biol. 2012;108:367-93 - PubMed
  5. Sensors (Basel). 2017 Mar 03;17(3): - PubMed
  6. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3795-9 - PubMed
  7. J Hepatol. 2021 Jun;74(6):1429-1441 - PubMed
  8. J Clin Invest. 2002 Sep;110(5):583-90 - PubMed
  9. Toxins (Basel). 2016 Mar 08;8(3): - PubMed
  10. Anal Biochem. 1969 Mar;27(3):502-22 - PubMed
  11. Hepatology. 2014 Sep;60(3):779-81 - PubMed
  12. Nat Methods. 2012 Jul;9(7):671-5 - PubMed
  13. Front Neurol. 2019 Nov 07;10:1168 - PubMed
  14. Redox Biol. 2017 Apr;11:60-72 - PubMed
  15. J Lipid Res. 2016 Feb;57(2):299-309 - PubMed
  16. Cancers (Basel). 2021 Aug 13;13(16): - PubMed
  17. Can J Physiol Pharmacol. 2017 Aug;95(8):894-903 - PubMed
  18. J Neurosci. 2009 May 20;29(20):6394-405 - PubMed
  19. J Lipid Res. 2011 Jul;52(7):1345-51 - PubMed
  20. Mol Genet Metab. 2012 Jul;106(3):330-44 - PubMed
  21. iScience. 2020 Oct 15;23(11):101691 - PubMed
  22. Protein Eng. 2001 Dec;14(12):993-1000 - PubMed
  23. Cell. 2010 Jan 22;140(2):197-208 - PubMed
  24. Methods Enzymol. 1987;143:101-9 - PubMed
  25. Mol Membr Biol. 2007 Mar-Apr;24(2):121-34 - PubMed
  26. Orphanet J Rare Dis. 2014 Apr 28;9:64 - PubMed

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