title: A simple enrichment procedure improves detection of membrane proteins by immunoblotting.
abstract: We developed a novel approach to improve detection of membrane-associated proteins in yeast cell lysates by immunoblotting. Our method consists of a simple enrichment procedure using sedimentation to remove soluble proteins and the use of an alternative electrophoresis sample buffer, which allows for protein solubilization without heating. The efficacy of this approach was demonstrated for membrane proteins inHansenula polymorpha(Pho87, Gas1, and Pmr1) andSaccharomyces cerevisiae(Gas1). Immunoblot analysis of proteins that are not membrane-associated showed that the precipitate fraction was depleted of Sup45, carboxypeptidase Y, and Hog1; however, tubulin and, to some extent, Sup35 and Tpd3 were precipitated together with the membrane proteins. The presence of tubulin in the same fraction as the membrane proteins allows its use as a reference protein.
authors.affiliation: Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences.
authors.forename: Azamat
authors.initials: A
authors.lastname: Karginov
authors.affiliation: Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences.
authors.forename: Michael
authors.initials: M
authors.lastname: Agaphonov
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D015151:Immunoblotting / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010843:Pichia / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins
keywords: gel electrophoresis; immunoblotting; membrane protein; protein extraction; yeast