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Open Access Highly Accessed Methodology article

Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition

Ritchie Williamson12*, Andrew J Thompson1, Mika Abu1, Abdul Hye1, Alessia Usardi1, Steven Lynham1, Brian H Anderton1 and Diane P Hanger1

Author Affiliations

1 MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, London, UK

2 Biomedical Research Institute, University of Dundee, Ninewells Medical School, Dundee, UK

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BMC Neuroscience 2010, 11:120  doi:10.1186/1471-2202-11-120

Published: 22 September 2010

Abstract

Background

Membrane rafts are small highly dynamic sterol- and sphingolipid-enriched membrane domains that have received considerable attention due to their role in diverse cellular functions. More recently the involvement of membrane rafts in neuronal processes has been highlighted since these specialized membrane domains have been shown to be involved in synapse formation, neuronal polarity and neurodegeneration. Detergent resistance followed by gradient centrifugation is often used as first step in screening putative membrane raft components. Traditional methods of raft isolation employed the nonionic detergent Triton X100. However successful separation of raft from non-raft domains in cells is dependent on matching the detergent used for raft isolation to the specific tissue under investigation.

Results

We report here the isolation of membrane rafts from primary neuronal culture using a panel of different detergents that gave rise to membrane fractions that differed in respect to cholesterol and protein content. In addition, proteomic profiling of neuronal membrane rafts isolated with different detergents, Triton X100 and CHAPSO, revealed heterogeneity in their protein content.

Conclusions

These data demonstrate that appropriate selection of detergent for raft isolation is an important consideration for investigating raft protein composition of cultured neurons.