Semi dry electrophoresis

Sodium Dodecyl Sulfate–Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed following standard methods [67 ] using the Miniprotean III system (Bio-Rad). The sample buffer for reducing SDS-PAGE is 60 mM Tris–HCl pH 6.8, 1% w/v SDS, 5% v/v glycerol, 0.005% w/v bromophenol blue and 1% v/v 2-mercapto-ethanol (2-ME). In non-reducing SDS-PAGE, the sample buffer was devoid of 2-ME. Proteins separated by SDS-PAGE were either stained with Coomassie Blue R-250 (Bio-Rad) or subjected to Western blot. For the latter, the proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Immobilon-P, Millipore) using semi-dry electrophoresis (Bio-Rad), as previously described [67 ]. Nb-HlyA fusions were detected with primary mAb anti-E-tag (Phadia, 1:5000) and secondary polyclonal rabbit anti-mouse IgG antibodies fused to POD (1:5000, Sigma). Membranes were developed using a mixture of 100 mM Tris–HCl (pH 8.0) containing 1.25 mM luminol (Sigma), 0.22 mM cumaric acid (Sigma), and 0.0075% (v/v) H₂O₂ (Sigma). The membranes were then developed by exposure to X-ray films (AGFA).

Viral lysates or pelleted virus particles were mixed with 5× sample buffer and heated for 10 min at 98 °C before being subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a PVDF membrane (Roth, Karlsruhe, Germany) by semi-dry electrophoresis (Biorad, München, Germany). After blocking in PBS with 5% skim milk powder and 0.1% Tween, the membranes were probed with specific rat anti-CA sera as described previously [15 (link)] and a secondary horseradish peroxidase-conjugated goat-anti-rat antibody (Sigma-Aldrich, Hamburg, Germany). The β-actin was detected with the HRP-conjugated AC-40 mouse monoclonal antibody (Sigma-Aldrich). Proteins were visualized using SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA) and Kodak Medical X-ray film.