Assessment of SdrD Protein Expression

Expression of the SdrD protein was assessed by immunoblot analysis. Lysates of S. aureus subsp. aureus NCTC8325-4 and the isogenic NCTC8325-4 ΔsdrD mutant were prepared for Western blotting as described previously (4 (link)). To assess the release of SdrD, the culture supernatant was filter sterilized (pore size, 0.22 μm) to remove intact bacterial cells and concentrated using an Amicon Ultra 50K centrifugal filter device (Millipore Corp., USA). Expression of SdrD in the bacterial pellet and supernatant was evaluated by immunoblotting using SdrD A region-specific (a kind gift from Elisabet Josefsson) (primary) and polyclonal swine anti-rabbit immunoglobulin (Dako, Denmark) (secondary) antibodies.