Western Blot Detection of Hbl B

For Western blotting, 30 µl protein solution or B. cereus culture supernatant were applied to Criterion XT precast gels (BioRad Laboratories, Feldkirchen, Germany). After electrophoresis, proteins were blotted to a PVDF-P membrane (Millipore; Merck, Darmstadt, Germany). The membrane was saturated with 3% casein-PBS and incubated for 1 h at room temperature with 2 µg/ml Strep-MAB-Classic (IBA Lifesciences, Göttingen, Germany) or monoclonal antibody (mAb) 11A5 [21 (link)] for detection of Hbl B.’ Afterward, it was washed three times in PBS with 0.1% Tween 20 and incubated overnight with a 1:2000 dilution of rabbit anti-mouse-horseradish peroxidase conjugate (Dako; Merck, Darmstadt, Germany) before three further washing steps in PBS with 0.1% Tween 20 and two in PBS. Subsequently, Super Signal Western Femto (Pierce; Thermo Fisher Scientific, Waltham, MA, USA) was applied, and chemiluminescence signals were sensed on a UVP ChemStudio imager (Analytik Jena, Jena, Germany).