Western Blotting for Porphyromonas gingivalis Virulence Factors

Sample preparation, separation by SDS-PAGE, electro-blotting onto polyvinylidene difluoride or nitrocellulose membranes and blocking were performed as described previously53 (link). RgpA and RgpB catalytic domains were detected with rabbit polyclonal anti-Rgp antibodies; Kgp with mouse monoclonal anti-Kgp antibodies; PPAD with rabbit polyclonal anti-PPAD antibodies; and PorZ with mouse polyclonal anti-PorZ antibodies (see also above). In addition, oligohistidine-tagged PorZ was detected using mouse monoclonal THE^TM His-Tag antibodies (Genscript, USA) at 0.125 μg/ml. Development with polyclonal anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (BD Pharmingen and Amersham Pharmacia) was carried out using the ECL Western Blotting substrate kit according to the manufacturer’s instructions (Pierce, UK). Streptavidin conjugated to horseradish peroxidase was used to detect MmdC, a biotinylated IM-associated protein.