Fig. 2.

Pull-down assay of the endolysin PlyL C-terminal domain using live B. anthracis Sterne 34F₂ cells. The top panel shows a Coomassie-stained PAGE gel of the samples analyzed. Equal loading of B. anthracis cells was confirmed making use of the prominent ∼100 kDa band of the S-layer proteins Sap and EA1 (at 0 μM PlyL C-terminal domain, the equal number of B. anthracis cells was analyzed without PlyL C-terminal domain pretreatment). The western blot at the bottom of the figure shows the increasing amounts of the pulled-down PlyL C-terminal domain bound to B. anthracis cells after pretreatment with increasing concentrations of the PlyL C-terminal domain. For that, tubes containing a cell suspension/endolysin protein mixture were placed on a rocking platform and incubated for 10 min. After incubation, the cells were pelleted and resuspended in phosphate-buffered saline, washed and mixed with an equal volume of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) sample loading buffer, boiled and analyzed on SDS–PAGE gels. For western blots, the anti-His antibody and the Goat horseradish peroxidase conjugated anti-mouse antibody were used as the primary and the secondary antibodies, respectively (for more details, see Experimental).