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Generation of proteoliposomes from subcellular fractions
Author(s) -
Rojo Manuel,
Budin Nicolas,
Kellner Roland,
Gruenberg Jean
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181418
Subject(s) - integral membrane protein , membrane protein , membrane , peripheral membrane protein , lipid bilayer , chemistry , nanodisc , biochemistry , biophysics , chromatography , biology
Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent‐mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high‐resolution two‐dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl‐octaoxyethylene (C 12 E 8 ) and subsequent detergent removal with BioBeads SM‐2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.