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Isolation of Lipid Rafts Through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death Receptor Localization in Raft Fractions

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CD95

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1557))

Abstract

Lipid raft domains, enriched in sphingolipids and cholesterol, serve as sorting platforms and hubs for signal transduction proteins, and show resistance to detergent solubilization. Despite rafts have been involved in survival processes, these membrane domains have also been shown to play a major role in the modulation of death receptor signaling. Here, we describe a detailed protocol for isolating lipid rafts from whole cells by taking advantage of the lipid raft resistance to Triton X-100 solubilization at 4 °C, followed by sucrose gradient centrifugation, with subsequent analysis of Fas/CD95 death receptor localization in the raft fractions by immunoblotting. This method is also useful to localize additional proteins in membrane rafts.

The original version of this chapter was revised. The erratum to this chapter is available at: DOI 10.1007/978-1-4939-6780-3_22

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-4939-6780-3_22

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Acknowledgments

This work was supported in part by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R), the Instituto de Salud Carlos III (RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, cofunded by the European Regional Development Fund, ERDF, of the European Union), and the European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS).

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Authors and Affiliations

  1. Laboratory of Cell Death and Cancer Therapy, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), C/ Ramiro de Maeztu 9, 28040, Madrid, Spain

    Consuelo Gajate & Faustino Mollinedo

Authors
  1. Consuelo Gajate
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  2. Faustino Mollinedo
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Correspondence to Consuelo Gajate or Faustino Mollinedo .

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Editors and Affiliations

  1. ER440-OSS, Label INSERM, Universite de Rennes CLCC Eugene Marquis, RENNES, France

    Patrick Legembre

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Gajate, C., Mollinedo, F. (2017). Isolation of Lipid Rafts Through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death Receptor Localization in Raft Fractions. In: Legembre, P. (eds) CD95. Methods in Molecular Biology, vol 1557. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6780-3_13

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  • DOI: https://doi.org/10.1007/978-1-4939-6780-3_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6778-0

  • Online ISBN: 978-1-4939-6780-3

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