Browsing by Subject "2-DE"

The literature review describes the characteristics, uses and probiotic potential of propionibacteria and gives an overview of proteomic methods focusing on 2-DE studies related to probiotic bacteria and P. freudenreichii. The aim of the experimental part was to use 2-DE to obtain proteome maps of two strains of P. freudenreichii and detect inter-strain differential protein expression. The influence of a number of parameters on the quality of 2-D gels was also investigated. The pH 3-7 and pH 3-11 NL proteome maps obtained displayed 305 and 356 spots, respectively, and detected the differential expression of 60 and 72 spots, respectively. The 3-11 NL proteome map showed spot over-crowding in the pH 4-6 region. Addition of 50 mM dithiothreitol (DTT) to the samples prior to isoelectric focusing (IEF) and an increase of IPG buffer concentration greatly improved spot resolution. Silver staining achieved higher spot counts than Coomassie staining. The influence of the number of lysis cycles on protein extraction was marginal, while lower protein sample loads produced superior 2-D gels. Results show that data from 2-DE proteome maps can provide an overview of inter-strain differential protein expression in P. freudenreichii. The optimal 2-DE experimental setup would include pH 4-7 IPG strips, a low protein load and inclusion of DTT in the IEF sample, as well as silver staining for spot visualisation.