Rabbit anti np polyclonal antibody

Culture media from NoV-M2/NP baculovirus-infected Sf9 insect cells was harvested 96 h post-infection. Culture media from cells infected with an empty NoV platform was used as a control. Samples were loaded on 10%–20% precast WedgeWell Gel (Thermo Fisher Scientific, Waltham, USA) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto nitrocellulose membranes was performed using Trans-Blot Turbo RTA Mini 0.2 μm Nitrocellulose Transfer Kit (Bio-Rad, Hercules, USA). Membranes were then blocked for 1 h in a 5% semi-skimmed milk solution (5% milk/TBS/0.01% Tween 20) and incubated overnight with primary antibodies solution: rabbit polyclonal anti-NoV serum (obtained by vaccination of rabbit with insect cell-derived NoV VP1), mouse monoclonal anti-M2 (Santa Cruz Biotechnologies, Dallas, USA) rabbit anti-NP polyclonal antibody (Genetex, Irvine, USA), or anti-β-actin antibodies (Abcam, Waltham, USA) in 5% milk/TBS/0.01% Tween 20. Then, the membranes were washed (TBS/0.01% Tween 20) and incubated with a solution of AffiniPure goat anti-rabbit or anti-mouse antibodies (Jackson Immuno Research, Ely, United Kingdom) in 5% milk/TBS/0.01% Tween 20. The reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, USA).