Proteolytic artifacts in SDS‐PAGE analysis of selected periodontal pathogen:

摘要: The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution protein bands, occur during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis cellular proteins from selected (Porphyromonas gingivalis, Prevotella nigrescens Treponema denticola) non-proteolytic (Fusobacterium nucleatum) bacteria. Conditions limit or prevent proteolysis were also investigated. Bacterial cells incubated solubilizing buffer (SDS+ beta mercaptoethanol) at room temperature for various periods time before boiling. A control assay consisted trichloroacetic acid-treated bacterial cells. Cellular separated by stained with Coomassie blue. Proteolysis occurred very rapidly case P. gingivalis ( 1 h) required observe similar effects T. denticola. No observed F. nucleatum. In all cases, heat (100 degrees C) low pH (< 4) treatments could avoid production artifacts. Incorporation specific protease inhibitors solubilization bacteria proteolysis. More particularly, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetamide diisopropylfluorophosphate (50 mM) highly efficient denticola, respectively. When outer membranes prepared presence TLCK, numerous additional brands, not seen absence detected. present suggests that inhibitors, effective preventing proteolysis, should be identified added cell fractionation purification procedures.