Проблемы особо опасных инфекций (Mar 2016)
Application of 2-D Electrophoresis for the Construction of “Protein Profiles” of Bacterial Cultures Lysates of Particularly Dangerous Infections Agents
Abstract
Objective of the study is to deploy 2-D electrophoresis for the construction of lysate “protein profiles” of bacterial cultures of particularly dangerous infections. Materials and methods. “Protein profiles” are obtained on the model of non-toxigenic V. cholerae biovar El Tor M-888 strain and Y. pestis EV NIIEG strain, cultivated at 28 and 37 °C. Commercial sample of E. coli strain lysate serves as comparator (reference) product. SDS-PAGE-electrophoresis is carried out following classical Laemmli approach. 2D-electrophoresis is based on O’Farrel technique and manufacturer’s recommendations. For protein visualization, staining of electrophoregrams with coomassie blue G-250 and silver nitrate is used. Protein concentration (load) is evaluated with the help of Bradford protein assay. Cell lysate samples are purged of non-protein impurities applying Ready Prep 2-D Cleanup Kit. Results and conclusions. Described is a highly effective complex method of protein mixture separation, which assumes preparation of bacterial culture probes where cells are fractured under ultrasonic exposure in the lysing buffer, then purified and analyzed either in reference to the charge in pH gradient, or - to molecular mass in SDS-PAGE-electrophoresis. After the staining 2-D gels are assayed by means of “Dymension” software product, installed on the platform of multi-functional gel documentation system “Syngene”. Utilization of stripes with immobilized pH gradient of a preset range, commercial reagent kits, and large-sized polyacrylamide gels has allowed for optimization of the conditions for performing 2D-electrophoresis, reduction of the time elapsed, and improvement of accuracy and reproducibility. On the model of non-toxigenic V. cholerae biovar El Tor M-888 strain and Y. pestis EV NIIEG strain, cultivated at 28 and 37 °C, applying 2D-electrophoresis, obtained are the protein “profiles” of the investigated strains, demonstrated is high efficacy of the method and possibility of its deployment for studies of gene expression dynamics as regards agents of particularly dangerous infections.
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