Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition.
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.
Williamson
Ritchie
R
MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, UK. R.Williamson@dundee.ac.uk
Thompson
Andrew J
AJ
Abu
Mika
M
Hye
Abdul
A
Usardi
Alessia
A
Lynham
Steven
S
Anderton
Brian H
BH
Hanger
Diane P
DP
D000818:Animals; D015153:Blotting, Western; D037281:Calnexin / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry* / Q000187:drug effects; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D040901:Proteomics; D051381:Rats; D011990:Receptors, Transferrin / Q000737:chemistry / Q000378:metabolism
D003902:Detergents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011990:Receptors, Transferrin; C106354:flotillins; D037281:Calnexin; D011092:Polyethylene Glycols; D002784:Cholesterol