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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Mol Microbiol. 2010 Dec 22;79(4):954–967. doi: 10.1111/j.1365-2958.2010.07505.x

Fig. 2. Analysis of the PapC N-terminal disulfide loop mutants.

Fig. 2

A. Overlay assay for binding of PapDG to PapC. OM fractions were isolated from strain SF100 expressing vector only, WT PapC, or the indicated PapC mutant. Duplicate samples were subjected to SDS-PAGE and either stained with Coomassie blue (upper panel) or transferred to a PVDF membrane for the overlay assay. Binding of PapDG to the usher (lower panel) was determined by immunoblotting with anti-PapDG antibodies.

B. Co-purification of pilus tip subunits with PapC. OM fractions were isolated from strain SF100/pPAP58 (papDJKEFG) expressing vector only, WT PapC, or the indicated PapC mutant. His-tagged PapC was purified from the OM fractions, subjected to SDS-PAGE, and either stained with Coomassie blue to show the amount of PapC present (upper panel) or immunoblotted with anti-P pilus tips antiserum to detect pilus tip subunits that co-purified with the usher (PapG, K, E and F, lower panel).

C. Assembly of PapG into pilus tip fibers. Co-purification of pilus tip subunits with WT PapC or the indicated PapC mutant was performed as in (B). The samples were incubated at 25 or 95°C in SDS-PAGE sample buffer, subjected to SDS-PAGE and immunoblotted with anti-PapDG antibodies.