title: 16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.
abstract: Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-BAC and SDS, respectively. Mitochondria were isolated from yeast cultures by differential centrifugation and were further purified by free flow electrophoresis (FFE) in zone-electrophoretic mode (ZE). Subsequently, membrane proteins from ZE-FFE-purified mitochondria were enriched by carbonate extraction and subjected to 16-BAC/SDS-PAGE. The resulting protein spot patterns were visualized by a highly sensitive fluorescence stain with ruthenium-II-bathophenantroline disulfonate chelate (RuBP), and by colloidal Coomassie staining. Proteins were identified by Maldi-Tof mass spectrometry and peptide mass fingerprinting.
authors.affiliation: GSF-National Research Centre for Environment and Health, Institute of Human Genetics, Munich, Germany.
authors.forename: Ralf J
authors.initials: RJ
authors.lastname: Braun
authors.forename: Norbert
authors.initials: N
authors.lastname: Kinkl
authors.forename: Hans
authors.initials: H
authors.lastname: Zischka
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
mesh_terms: D002254:Carbonates / Q000737:chemistry; D002498:Centrifugation; D005591:Chemical Fractionation / Q000379:methods*; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005233:Fatty Alcohols; D005456:Fluorescent Dyes; D008565:Membrane Proteins / Q000032:analysis*; D008928:Mitochondria / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012428:Ruthenium / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D002254:Carbonates; D005233:Fatty Alcohols; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; D012967:Sodium Dodecyl Sulfate; C005686:sodium carbonate; D012428:Ruthenium; C012817:cetalkonium chloride