Analysis of bacterial proteins by 2DE.

Two-dimensional gel electrophoresis (2DE) is a key analytical method for investigating bacterial -proteomes. The relatively simple genomes of many bacteria combined with only limited post--translational modifications of bacterial proteins mean that a significant proportion of the proteome is open to analysis by 2DE. The applications of 2DE in the field of microbiology are diverse and range from analysing physiological responses of the bacteria to environmental stress to investigating bacterial pathogenesis in human bacterial pathogens. The standard approach for 2DE in the analysis of bacterial proteins uses immobilised pH gradient (IPG) gels in the first dimension for charge separation and then an orthogonal separation, in the presence of SDS, to resolve the proteins according to their molecular mass. Protocols are presented in this chapter for small (7-cm-length IPG gel strips)- and medium (11- or 13-cm-length IPG strips)-format 2D gels using IPG gels and SDS-containing polyacrylamide slab gels for the second dimension. The application of the methods are demonstrated for the analysis of cell lysates prepared from Helicobacter pylori, although the same protocols have been used to analyse proteins from a variety of human bacterial pathogens.