HPV16 L1 Protein Immunoblot Detection

Equal volume aliquots from organotypic tissue extracts were boiled for 10 minutes in 6% 2 mercaptoethanol (2-ME) loading buffer and loaded onto a 7.5% polyacrylamide gel. Nitrocellulose membranes were blocked using StartingBlock blocking buffer in PBST (Thermo Scientific). To detect HPV16 L1, the anti-HPV16 L1 monoclonal antibody Camvir-1 (BD Pharmigen) was utilized at a 1∶2,000 dilution. An HRP-linked sheep anti-mouse secondary antibody was utilized at a 1∶8,000 dilution. Membranes were washed with PBST after the addition of each antibody. All antibodies were diluted in StartingBlock (Thermo Scientific). HRP was detected using an ECL kit (Perkin Elmer). The conditions for non-reducing L1 immunoblots have been previously described [29] (link). Results are representative of at least 2 different batches of virus preps for each virus type.