Visualisation of detergent solubilisation of membranes: Implications for the isolation of rafts
Ashley E Garner 1, Alastair Smith 1 and Nigel M. Hooper 1*
1 University of Leeds
* To whom correspondence should be addressed. E-mail: n.m.hooper{at}leeds.ac.uk.
Submitted on June 1, 2007
Revised on July 5, 2007
Accepted on 17 September 2007
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Abstract |
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Although different detergents can give rise to detergent resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and non-raft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilisation of the cell membrane and that of the supported lipid bilayers. In both systems Triton X100 and CHAPS distinguished between the non-raft ld and raft lo lipid phases by selectively solubilising the ld phase. A higher concentration of Lubrol was required and not all the ld phase was solubilised. The solubilisation by Brij 96 occurred by a two stage mechanism that initially resulted in the solubilisation of some ld phase and then progressed to the solubilisation of both ld and lo phases simultaneously. Octyl glucoside simultaneously solubilised both lo and ld phases. These data show that the mechanism of membrane solubilisation is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems
Key Words:
Triton X-100, atomic force microscopy, immunofluorescence microscopy, membrane raft, supported lipid bilayer
