engleski

Lipid raft isolation from mouse brain under conditions that retain ganglioside distribution

Lipid rafts are dynamic membrane microdomains with distinct lipid and protein compositions. They serve as signalling platforms of the cell and are involved in regulation of membrane functions. The established role of lipid rafts in pathogenesis of numerous human disorders has led to increased interest in investigation of lipid rafts dynamics. There are many methodological challenges in isolating lipid rafts, the major difficulty being their separation from the bulk (non-raft) membranes. Most often, detergent- resistant membranes (DRMs) enriched in cholesterol and sphingomyelin and thought to be related to rafts, are extracted using the non-ionic detergent Triton X- 100 (Tx-100). However, Tx- 100 causes a major redistribution of membrane gangliosides and is therefore unacceptable as a detergent for lipid raft isolation for studying gangliosides and ganglioside- associated proteins. The aim of this work was to optimize lipid raft isolation from mouse brain for the purposes of a larger study investigating the influence of membrane lipid composition on expression and distribution of selected proteins. In order to avoid ganglioside redistribution, detergents other than Tx-100 (saponin, Brij 98) and non- detergent methods were tested. Brij 98 was found to be appropriate, as a detergent that does not result in ganglioside redistribution, for generating DRMs that can be isolated by optimized sucrose density ultracentrifugation. Successful raft isolation was confirmed by Western blot analysis of the lipid raft marker protein flotilin and the non-lipid raft membrane protein transferrin receptor. Detection of gangliosides in raft and non-raft fractions was performed by immuno-overlay using anti-ganglioside antibodies as well as cholera toxin B subunit overlay. The developed method provides a tool to study lipid raft components and their changes under conditions that do not result in ganglioside redistribution.

Lipid rafts; gangliosides; Brij 98; Triton X-100

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