Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135–170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.
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Research Article|
May 15 2004
Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells Available to Purchase
Galina RADEVA;
Galina RADEVA
Department of Chemistry and Biochemistry, University of Guelph, Guelph, ON, Canada N1G 2W1
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Frances J. SHAROM
Frances J. SHAROM
1
Department of Chemistry and Biochemistry, University of Guelph, Guelph, ON, Canada N1G 2W1
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
September 03 2003
Revision Received:
February 03 2004
Accepted:
February 09 2004
Accepted Manuscript online:
February 09 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 380 (1): 219–230.
Article history
Received:
September 03 2003
Revision Received:
February 03 2004
Accepted:
February 09 2004
Accepted Manuscript online:
February 09 2004
Citation
Galina RADEVA, Frances J. SHAROM; Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells. Biochem J 15 May 2004; 380 (1): 219–230. doi: https://doi.org/10.1042/bj20031348
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