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Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence Microscopy: The Use of Combined Techniques to Assess Fas/CD95 Location in Rafts During Apoptosis Triggering

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Lipid Rafts

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2187))

Abstract

Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.

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Acknowledgments

This work was supported by Spanish Ministry of Science, Innovation and Universities grant SAF2017-89672-R.

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Authors and Affiliations

  1. Laboratory of Cell Death and Cancer Therapy, Department of Molecular Biomedicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain

    Consuelo Gajate & Faustino Mollinedo

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

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Editors and Affiliations

  1. Department of Physiology, University of Kentucky Medical School, Lexington, KY, USA

    Erhard Bieberich

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Gajate, C., Mollinedo, F. (2021). Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence Microscopy: The Use of Combined Techniques to Assess Fas/CD95 Location in Rafts During Apoptosis Triggering. In: Bieberich, E. (eds) Lipid Rafts. Methods in Molecular Biology, vol 2187. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0814-2_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0814-2_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0813-5

  • Online ISBN: 978-1-0716-0814-2

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