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

An improved protocol for the two-dimensional analysis of proteins of the Co rynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CH APS) concentrations (2-4%) and an optimized electrophoresis protocol, horiz ontal 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 IPG-phor isoelectric focusing unit can be us ed for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, th e preparation and analysis of C, glutamicum cell surface proteins is descri bed. Proteins were extracted with lauroyl sarcosinate and 100-120 spots wer e separated on two-dimensional (2-D) gels in comparison to 18-20 spots obse rved previously by standard sodium dodecyl sulfate-polyacrylamide gel elect rophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into thr ee distinct fractions resembling different functional units of the bacteria l cell.