Protein synthesis elongation factor EF-Tu is presented as an internal calibration standard for quantitative analysis of two-dimensional (2-D) protein electrophoresis gels. EF-Tu was selected on the basis of concentration measurements in cell-free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one-dimensional (1-D) and 2-D electrophoresis gels. The results demonstrated that the amount of EF-Tu synthesized in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF-Tu an ideal internal protein standard for quantitation of protein spots on 2-D electrophoresis gels. For such quantitative analysis we developed a computer-aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.

Academy of Sciences of the Czech Republic Institute of Microbiology Prague

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Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis