Biotin labeled goat anti mouse igg

Western blot analysis was used to evaluate the specificity of mAbs towards recombinant CPE (aa 26–319, wild type) and CPE domains PFD (aa 26-202; GST-PFD-t-mCherry) and RBD (aa 203-319). For SDS-PAGE, the antigens were mixed with 3 × Laemmli loading buffer (150 mM Tris-HCl, pH 6.8, 6% SDS, 30% (v/v) glycerol, 7.5% (v/v) β-mercaptoethanol, 0.25% (w/v) bromophenol blue) and heated for 10 min at 95 °C. After cooling on ice, samples were loaded onto 12% polyacrylamide gels and separated electrophoretically according to standard procedures [80 (link)]. Subsequently, gels were transferred to methanol activated Immobilon® P 0.45 μm PVDF membranes (Millipore, Schwalbach, Germany). Membranes were blocked at 4 °C overnight in 2% skimmed milk in PBS-T and incubated for 1 h at room temperature with 1 μg/mL of CPE-specific antibodies diluted in blocking buffer. After three washing steps, detection was performed by biotin-labeled goat anti-mouse IgG (Dianova, Hamburg, Germany) diluted 1:10,000 in blocking buffer for 30 min at room temperature. Membranes were developed using alkaline phosphatase-conjugated streptavidin (Avidix™-AP, Fisher Scientific, Bremen, Germany) and CDP-Star as substrate (Perkin Elmer, Waltham, MA, USA). Development was documented on a ChemiDoc imaging system (Bio Rad Laboratories, Hercules, CA, USA).

In total, 100 ng abrin, APA, ricin or BSA were separated on a 12% SDS-PAGE under reducing conditions and transferred onto an Immuno-Blot 0.45 μm PVDF membrane (Invitrogen, Karlsruhe, Germany). After blocking the membrane in blocking buffer (2% skimmed milk in PBST) at 4 °C overnight, diluted primary anti abrin/APA antibodies (final concentration 5 µg/mL) in blocking buffer were added to the membrane for 1 h. After three washing steps, the membrane was incubated with biotin-labeled goat anti-mouse IgG (1:5000; Dianova, Hamburg, Germany; for detection of mouse mAbs) or biotin-labeled goat anti-rabbit IgG (1:5000; Dianova, Hamburg, Germany; for detection of polyclonal antibody KAP142) in blocking buffer at room temperature for 30 min and was developed with avidin–alkaline phosphatase (incubation for 20 min, final concentration 0.5 µg/mL in PBST; Avidx™-AP, Fisher Scientific, Bremen, Germany) and the chemiluminescent substrate CDP-Star (Perkin Elmer, Waltham, MA, USA). Images were captured by a CCD camera (ChemiDoc, BioRad, Feldkirchen, Germany).