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Two‐dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins
Author(s) -
Hermann Thomas,
Finkemeier Melanie,
Pfefferle Walter,
Wersch Gregor,
Krämer Reinhard,
Burkovski Andreas
Publication year - 2000
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/(sici)1522-2683(20000201)21:3<654::aid-elps654>3.0.co;2-1
Subject(s) - corynebacterium glutamicum , isoelectric focusing , chaps , chromatography , membrane protein , gel electrophoresis , electrophoresis , polyacrylamide gel electrophoresis , sodium dodecyl sulfate , chemistry , membrane , cytoplasm , biochemistry , biology , gene , enzyme
An improved protocol for the two‐dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) concentrations (2—4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPGphor isoelectric focusing unit can be used for the in‐gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100—120 spots were separated on two‐dimensional (2‐D) gels in comparison to 18—20 spots observed previously by standard sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell.