Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins

• Published in: Electrophoresis Vol. 21; no. 3; pp. 654 - 659
• Main Authors: Hermann, Thomas, Finkemeier, Melanie, Pfefferle, Walter, Wersch, Gregor, Krämer, Reinhard, Burkovski, Andreas
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.02.2000
• Subjects: Amino Acid Sequence; Bacterial Proteins - analysis; Bacterial Proteins - isolation & purification; Blotting, Western; Corynebacterium - chemistry; Corynebacterium glutamicum; Electrophoresis, Gel, Two-Dimensional - methods; Membrane proteins; Membrane Proteins - analysis; Membrane Proteins - isolation & purification; Molecular Sequence Data; Proteomics; Surface proteins; Two-dimensional polyacrylamide gel electrophoresis
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/(SICI)1522-2683(20000201)21:3<654::AID-ELPS654>3.0.CO;2-1

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.