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dc.contributor.authorGajate, Consueloes_ES
dc.contributor.authorMollinedo, Faustinoes_ES
dc.identifier.citationLipid Rafts. Methods in Molecular Biology 2187:147-186 (2021)es_ES
dc.description40 p.-11 fig.-6 tab.es_ES
dc.description.abstractLipid 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.es_ES
dc.description.sponsorshipThis work was supported by Spanish Ministry of Science, Innovation and Universities grant SAF2017-89672-R.es_ES
dc.publisherSpringer Naturees_ES
dc.subjectLipid raftses_ES
dc.subjectCell disruptiones_ES
dc.subjectPotter-Elvehjem homogenizeres_ES
dc.subjectDetergent-resistant membraneses_ES
dc.subjectTriton X-100–resistant membraneses_ES
dc.subjectSucrose gradient centrifugationes_ES
dc.subjectLipid raft isolationes_ES
dc.subjectGel electrophoresises_ES
dc.subjectFluorescence microscopyes_ES
dc.subjectCholera toxin B subunites_ES
dc.titleLipid 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 triggeringes_ES
dc.typecapítulo de libroes_ES
dc.description.peerreviewedPeer reviewedes_ES
dc.contributor.funderMinisterio de Ciencia, Innovación y Universidades (España)es_ES
oprm.item.hasRevisionno ko 0 false*
dc.contributor.orcidGajate, Consuelo [0000-0003-0604-6459]es_ES
dc.contributor.orcidMollinedo, Faustino [0000-0002-4939-2434]es_ES
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