The optimal conditions for protein analysis of Treponema pallidum subsp. pallidum by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Sample solubilization techniques can influence the protein patterns obtained by SDS-PAGE. In this study we determined the effects of the amount of SDS present in the sample solubilization buffer on one and two dimensional (1D and 2D) electrophoretic protein profiles of radiolabeled T. pallidum as well as the antigenicity of treponemal proteins as judged by Western blots of 1D and 2D gels probed with pooled secondary human syphilitic sera. Although the total number of protein bands in 1D profiles obtained at SDS concentrations between 0-5% was not significantly different, there was an obvious shift in the location of the bands. However, a significant amount of variation (including isoelectric point and molecular weight shifts) was observed among the total number of protein species in 2D profiles. The greatest number of protein species (158 total) was obtained with 3% SDS in the solubilization buffer, followed by the 1% SDS giving raise to 143 protein species. The highest number of proteins (59 total) detected on Western blots of 2D profiles was obtained when no SDS was used. The number of antigenic proteins in 1D profiles followed a bell shaped curve with 2% SDS giving the highest number (32 total) of the antigenic proteins. With different concentrations of SDS, the number of antigenic proteins in 2D profiles decreased as the concentration of SDS increased. Thus, we conclude that, 2% SDS in the sample solubilization buffer is best for 1D profiles. However, SDS is not recommended to separate all of the antigenic proteins in 2D gels.
Sayahtaheri
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Department of Microbiology and Immunology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Cox
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D001345:Autoradiography; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007124:Immunoenzyme Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D012995:Solubility; D014210:Treponema pallidum / Q000032:analysis*
D011506:Proteins