Affinity purified horseradish peroxidase hrp conjugated anti mouse igg

The expression of recombinant L. lactis/pNZ8008-NP was detected by Western blot analysis as described previously [36 (link)]. In a brief, 5 × 10⁵ cells of L. lactis/pNZ8008-NP pellets were mixed with 60 µl of 6 × loading buffer and boiled for 10 min, then run on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, California, USA). The membrane was blocked with 5% non-fat milk, and then incubated with a 1:500 polyclonal mouse anti-NP antibody (kindly provided by NIH Biodefense and Emerging Infections Research Resources Repository, Manassas, VA, USA), overnight at 4°C. Affinity-purified horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma-Aldrich Corporation, St. Louis, MO, USA) was used as second antibody. Finally, the proteins were visualized using enhanced chemiluminescence (ECL) reagents (GE Healthcare) according to the manufacturer’s instructions.

The expression of recombinant L. lactis/pNZ8149-HA1-M2 was determined by Western blot analysis. Briefly, 5 × 10⁵ cells of L. lactis/pNZ8149-HA1-M2 cells were mixed with 60 μl of 6 × loading buffer and boiled for 10 min, after which the proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, California, USA). The membranes were blocked with 5% skim milk and then incubated with a 1:500-diluted monoclonal mouse anti-HA or anti-M2 antibody (kindly provided by Beiresources, Manassas, VA, USA) overnight at 4 °C. Affinity-purified horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma-Aldrich Corporation, St. Louis, MO, USA) was used as the secondary antibody. Finally, the membrane was visualized using enhanced chemiluminescence reagents (GE Healthcare) according to the manufacturer’s instructions. Western blot analysis was performed by three independent experiments.