Hrp conjugated mouse monoclonal anti his antibody

Aliquots of cell-free extracts (15 µg of membrane protein extract for PBP2a or 10 µg of total protein for PrsA and HtrA1) were resolved in 10% SDS PAGE gels and transferred to Porablot NCP nitrocellulose membranes (0.45 µm; Machery-Nagel, Switzerland) by semi-dry transfer (Bio-Rad). Membranes were stained with Ponceau Red and photographed to assure transfer uniformity. Membranes were blocked with non-fat milk 5% w/v in PBS-0.2 % Tween-20 washed in PBS/Tween. Proteins were detected using respectively: mouse monoclonal anti-PBP2a (bioMérieux France, Slidex MRSA detection kit, ref 73117) (dilution 1:500), rabbit anti-PrsA^{20 (link)} (dilution 1:50,000) or rabbit anti-HtrA1 (Eurogentec, Belgium, custom anti-peptide antibody: KLH-conjugated C + TNNKGGNQLDGQSKK) dilution 1:20,000). Secondary antibodies, HRP-conjugated goat anti-mouse or anti-rabbit Ig (Bio-Rad) were diluted 1:5000 in PBS-Tween. His-tag purified PBP2a was detected using HRP-conjugated mouse monoclonal anti-HIS antibody (Invitrogen). HRP-conjugated mouse monoclonal anti-HIS antibody (Invitrogen) was used at 1:500. Blots were washed and developed using West Pico enhanced chemiluminesence according to the manufacturer’s recommendations (Pierce).