Analyzing Protein Expression in Modified Adenoviral Vectors

Protein expression was analyzed from lysates of HEK293 cells infected with Ad5-CMV-ASP-2 and Ad48-CMV-ASP-2. The other modified vectors, Ad5-pIX-ASP-C, Ad5-pIX-gp83, Ad48-pIX-ASP-C, and Ad48-pIX-gp83 (5 × 10⁹ VPs/vector) were analyzed for the protein of interest incorporated within the capsid. The lysates and vectors were denatured by boiling and resolved on SDS-PAGE gels, followed by transfer onto polyvinylidene difluoride (PVDF) membranes, which were then blocked with 5% dry non-fat milk in PBST for 1 h. Thereafter, the membranes were incubated overnight at 4 °C with His₆ MAb (1:5000 dilution in blocking buffer; GenScript), or FLAG-HRP antibody. After overnight incubation, the membranes were washed, and the His₆ membrane was incubated with HRP-conjugated goat anti-mouse antibody (1:5000; Millipore, Temecula, MA, USA). The proteins were detected by using 3′3′-diaminobenzidine tablets (Sigma-Aldrich, St. Louis, MO, USA) [56 (link)].