Pvdf p membrane

SDS-PAGE analyses were carried out on a PhastGel gradient (10 to 15%) minigel system (GE Healthcare, Munich, Germany). For Sypro staining, proteins were fixed on the gel for 2 × 30 min in 50% MeOH and 7% acetic acid. The gel was then incubated with 2 mL Sypro Ruby protein stain overnight at room temperature. After 30 min washing in 10% MeOH and 7% acetic acid and additional washing for 10 min in H2O, fluorescence signals were detected on a Kodak imager (Eastman Kodak Company, Rochester, NY, USA).
For immunoblotting, proteins were blotted to a PVDF-P membrane (Millipore, Billerica, MA, USA), blocked in 3% casein-PBS and incubated with 2 μg/mL mAbs 8B12 (Hbl L2) [32 (link)], 1E9 (Hbl L1) [29 (link)], 1B8 (Hbl B) [29 (link)], 1E11 or 2B11 (NheB) [20 (link)], 1A8 (NheA) [20 (link)], 3D6 (NheC) [21 (link)], mAb 1H9 (this study), or the strep-specific StrepMAB-Classic (iba lifesciences) for 1 h at room temperature. After 3 washing steps in PBS with 0.1% Tween 20, a 1:2000 dilution of rabbit anti-mouse-horseradish peroxidase conjugate (Dako) was used as secondary antibody. After three further washing steps in PBS with 0.1% Tween 20 and two in PBS, Super Signal Western Femto Maximum Sensitivity Substrate (Pierce) was applied. Chemiluminescence signals were detected on a Kodak imager (Eastman Kodak).

SDS-PAGE was performed on Novex^TM WedgeWell^TM 8–16% Tris-Glycine gels (Invitrogen; Thermo Fisher Scientific, Waltham, USA) for 90 min at 125 V. After electrophoresis, proteins were blotted to a PVDF-P membrane (Millipore; Merck, Darmstadt, Germany), which was blocked in 3% casein-PBS and incubated for 1 h at room temperature with 2 μg/ml mAb 1A11 or with the rabbit antiserum (1:1000). The membrane was washed three times in PBS with 0.1% Tween 20 and incubated overnight with rabbit anti-mouse- or goat anti-rabbit-HRP (horseradish peroxidase) conjugate (Dako; Merck, Darmstadt, Germany) diluted 1:2000 in 1% casein-PBS. After three further washing steps in PBS with 0.1% Tween 20 and two in PBS, Super Signal Western Femto (Pierce; Thermo Fisher Scientific, Waltham, MA, USA) was applied and chemiluminescence signals were detected on a UVP ChemStudio imager (Analytik Jena, Jena, Germany).

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).