I am isolating lipid raft from animal tissue and cancer cell. How can I determine layer/layers in which lipid raft is present? I am using 5%, 40% and 80% sucrose gradient ultracentrifugation.
Isolation of Lipid Rafts Through Discontinuous Sucrose Gradient Centrifugation and Fas/CD95 Death Receptor Localization in Raft Fractions.
- Consuelo GajateEmail author
- Faustino MollinedoEmai
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.
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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