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• W2891006829 startingPage "2086" @default.
• W2891006829 abstract "Freely suspended liposomes are widely used as model membranes for studying lipid–lipid and protein–lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet’s lipid composition: the overall lipid composition is determined by gas chromatography–mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr3+. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies. Improved and robust procedures for a cyclodextrin-mediated preparation of asymmetric large unilamellar vesicles of diverse lipid compositions and the characterization of their degree of asymmetry and individual leaflet compositions with NMR and GC." @default.
• W2891006829 created "2018-09-27" @default.
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• W2891006829 date "2018-09-01" @default.
• W2891006829 modified "2023-10-11" @default.
• W2891006829 title "Preparation of asymmetric phospholipid vesicles for use as cell membrane models" @default.
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• W2891006829 doi "https://doi.org/10.1038/s41596-018-0033-6" @default.
• W2891006829 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6459367" @default.
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• W2891006829 hasPublicationYear "2018" @default.
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