|Smith, R - UNIVERSITY OF ILLINOIS|
Submitted to: American Society of Microbiologists Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: May 23, 2004
Publication Date: May 23, 2004
Citation: Smith, R.A., Kerr, B.J. 2004. Total protein and 2-d gel analysis of enterococcus faecalis proteins extracted by four methods. American Society of Microbiologists Abstracts. American Society of Microbiologists Abstracts. p.344. Technical Abstract: Both the identificatio of proteins and changes in levels of protein expression are important in determining the adaptation of Enterococcus faecalis to infection and stress. Two-dimensional electrophoresis is often used to make these determinations. Various chemical and mechanical methods are used to remove proteins from E. faecalis and little information is available concerning the qualitative and quantative differences in the proteins extracted by the different methods. The results presented here are from a comparative study of proteins obtained by four methods: two chemical (urea/thiourea detergent based extraction buffer and 1% SDS) and two mechanical (sonication and glass bead vortexing). Protein extracts were analyzed by determining total protein content and comparing 2-D electrophoresis gels of the isolated proteins using PDQuest gel analysis software (Bio-Rad). Sonication provided the largest quantity of total proteins extracted (4 mg/0.1 g of cells) while the least protein was recovered by solubilization in SDS (0.64 mg/0.1 g of cells). Although all four methods resulted in a similar number of spots on the gels (166 ± 4.8), sonication of the bacteria showed the best spot matching efficiency (87.6%) and the lowest variation in spot quantities (39% coefficient of variation). All methods showed a similar visual resolution of spots and comparable minimal streaking. Eleven percent of the 2-D gel spots from proteins isolated by glass bead vortexing were unique to that method. These unique proteins were not identified but they may be significant depending on the applicatio nof the 2-D gel analysis. Overall, quantitative and qualitative results were optimized when E. faecalis was sonicated as compared to mechanical lysis with glass beads or solubilization in SDS or extraction buffer.