Goat anti mouse antibody hrp

B. subtilis cells were grown until stationary phase in LB at 37°C, harvested by centrifugation and the pellet was resuspended in SSC prior to sonication. OD₆₀₀ was used to normalise the loading of samples in the SDS-PAGE gel. The gel was cut into two parts prior to transfer, and the upper portion was Coomassie stained to use as loading control and the lower portion was transferred to a PVDF membrane by electroblotting. Myc-tagged PcrA was detected using a monoclonal anti-myc antibody (Proteintech) followed by an anti-mouse-HRP goat antibody (Santa Cruz Biotechnology).

The proteins were separated depending on their molecular weight in SDS-PAGE using 12.5% acrylamide gels. The proteins were transferred on a nitrocellulose membrane using semi-dry blot chamber. Subsequent to Ponceau S (AppliChem GmbH, Darmstadt, Germany) staining, unspecific binding was blocked by incubation in 5% skim milk powder diluted in PBS-T (137 mM NaCl, 2.7 mM KCl 8 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Tween^®20, pH 7.4) for 1 h at RT. The membrane was quickly washed with PBS-T and incubated with actin monoclonal antibody (1:10,000, ACTN05(C4); Thermo Fisher Scientific, Waltham, MA, USA) or streptavidin-peroxidase conjugate (1:5000; Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS-T for 1 h at RT. Afterwards, unbound proteins were removed by washing three times with PBS-T for 5 min at RT on an orbital shaker. For the detection of actin, the membrane was further incubated with the secondary goat anti-mouse antibody-HRP (1:2500; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at RT and washed three times with PBS-T. The peroxidase marked proteins were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and X-ray films (AGFA Health Care, Mortsel, Belgium).