The eluted HEVD and HΔ200 proteins were separated on sodium dodecylsulfate (SDS)-8% polyacrylamide gels under denaturing conditions. The separated proteins were transferred onto nitrocellulose membranes (Bio-Rad Laboratories, USA) by electroblotting, and the membranes were incubated in TBS-Tween consisting of 25 mM Tris-HCl, 137 mM NaCl, 3 mM KCl, and 0.1% Tween 20 (pH 7.5) with 5% (v/v) skim milk (Sigma). After one 20-min wash and two additional 5-min washes, the membranes were incubated for 60 min at 20℃ with a monoclonal anti-His6 antibody (Roche, USA) diluted 1:2,000 in 1% bovine serum albumin (BSA; Sigma) and PBS. A similar Western blotting assay was also performed using goat anti-CDV polyclonal antibody (VMRD, USA) diluted 1:1,000 with 1% BSA in PBS. Antibody binding was detected by incubation with rabbit anti-goat-peroxidase conjugate (1:2,000; Pierce Biotechnology, USA) and peroxidase substrate (Pierce Biotechnology).
Independent samples of HEVD and HΔ200 (0.5 µg each) were also dot blotted onto single nylon membrane segments (Roche) using routine methods. The anti-CDV polyclonal antibody and anti-goat-horseradish peroxidase (HRP) antibody were used as the primary and secondary antibodies, respectively. Independent dot blots were also incubated with anti-His6-peroxidase antibody.
Independent samples of HEVD and HΔ200 (0.5 µg each) were also dot blotted onto single nylon membrane segments (Roche) using routine methods. The anti-CDV polyclonal antibody and anti-goat-horseradish peroxidase (HRP) antibody were used as the primary and secondary antibodies, respectively. Independent dot blots were also incubated with anti-His6-peroxidase antibody.