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2006 ; 332
(ä): 169-79
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Isolation of membrane rafts and signaling complexes
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Boesze-Battaglia K
Methods Mol Biol
2006[]; 332
(ä): 169-79
PMID16878692
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Traditionally, lipid rafts have been defined by their insolubility in ice-cold
Triton X-100 and low-buoyant density. These low-density membrane microdomains
have been referred to as detergent-resistant membranes, Triton-insoluble
membranes, and Triton-insoluble floating fraction. They are enriched in
cholesterol, often sphingomyelin and various gangliosides (GMI, GM2, and GM3).
The ability of the B-subunit of cholera toxin to bind GMI has been exploited to
visualize membrane rafts by confocal microscopy in patching and capping
experiments. Biochemically, membrane rafts are isolated by solubolization in
ice-cold Triton X-100 and separation of the low-buoyant density fractions from
soluble material on sucrose density gradients. We describe the isolation of
Jurkat cell-specific membrane rafts using 2% Triton X-100. This procedure yielded
a consistent raft product that was enriched in cholesterol, gangliosides
sphingomyelin and membrane raft protein markers including lck and lat 1.
Moreover, rafts were visualized using Alexa Fluor 647 cholera toxin capped with
anti-cholera toxin antibody. Co-localization of the C subunit of cytolethal
distending toxin to rafts was determined using patching techniques.
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