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Braun, R.J. ; Kinkl, N.* ; Zischka, H. ; Ueffing, M.

16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.

Methods Mol. Biol. 528, 83-107 (2009)
DOI
Open Access Green as soon as Postprint is submitted to ZB.
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.
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Publication type Article: Journal article
Document type Scientific Article
Editors Peirce, M.J.* ; Wait, R.*
Keywords mitochondria; saccharomyces erevisiae; 16-BAC; 16-BAC/SDS-PAGE; zone electrophoresis; free flow electrophoresis; ruthenium staining
ISSN (print) / ISBN 1064-3745
e-ISSN 1940-6029
Conference Title Membrane Proteomics: Methods and Protocols
Journal Methods in Molecular Biology
Quellenangaben Volume: 528, Issue: , Pages: 83-107 Article Number: , Supplement: ,
Publisher Springer
Publishing Place Berlin [u.a.]
Reviewing status Peer reviewed
Institute(s) CF Metabolomics & Proteomics (CF-MPC)
Institute of Molecular Toxicology and Pharmacology (TOX)