TY - JOUR
T1 - The isolation of detergent-resistant lipid rafts for two-dimensional electrophoresis.
AU - Kim, Ki Bum
AU - Lee, Jae Seon
AU - Ko, Young Gyu
PY - 2008
Y1 - 2008
N2 - Because lipid rafts are plasma membrane platforms mediating various cellular events such as in signal transduction, immunological response, pathogen invasion, and neurodegenerative diseases, protein identification in the rafts could provide important information to study their function. Here, we present an optimized method to isolate detergent-resistant lipid rafts that are subsequently analyzed by two-dimensional electrophoresis (2-DE). Lipid rafts were isolated based on their two distinct biochemical properties such as Triton X-100 insolubility and low density. To solubilize completely the proteins embedded in lipid rafts, sample lysis buffer (9 M urea, 2 M thiourea, 100 mM DTT, 2% CHAPS (w/v), 60 mM n-octylbeta-D-glucopyranoside, 2% IPG buffer) was applied to the isolated rafts. This method was found to be the most suitable choice for obtaining 2-DE profile of lipid raft proteome from various cells and tissues. We expect that this method could provide the way to dissect the function of raft-associated proteins and to gain a comprehensive insight upon various cellular events mediated through lipid rafts, the specialized domains in cell surface.
AB - Because lipid rafts are plasma membrane platforms mediating various cellular events such as in signal transduction, immunological response, pathogen invasion, and neurodegenerative diseases, protein identification in the rafts could provide important information to study their function. Here, we present an optimized method to isolate detergent-resistant lipid rafts that are subsequently analyzed by two-dimensional electrophoresis (2-DE). Lipid rafts were isolated based on their two distinct biochemical properties such as Triton X-100 insolubility and low density. To solubilize completely the proteins embedded in lipid rafts, sample lysis buffer (9 M urea, 2 M thiourea, 100 mM DTT, 2% CHAPS (w/v), 60 mM n-octylbeta-D-glucopyranoside, 2% IPG buffer) was applied to the isolated rafts. This method was found to be the most suitable choice for obtaining 2-DE profile of lipid raft proteome from various cells and tissues. We expect that this method could provide the way to dissect the function of raft-associated proteins and to gain a comprehensive insight upon various cellular events mediated through lipid rafts, the specialized domains in cell surface.
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U2 - 10.1007/978-1-60327-064-9_32
DO - 10.1007/978-1-60327-064-9_32
M3 - Review article
C2 - 18369879
AN - SCOPUS:44049084083
SN - 1064-3745
VL - 424
SP - 413
EP - 422
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -