S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen

Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander

Protein samples were heated in the SDS sample loading buffer (0.375 M Tris pH 6.8, 12 % SDS, 60 % glycerol, 0.6 M DTT, 0.06 % bromophenol blue) at 95°C for 5 min. Samples were separated by electrophoresis in 10 % SDS-PAGE followed by transfer of proteins onto nitrocellulose membrane in Towbin's buffer (0.025 M Tris, 0.192 M glycine, 20 % methanol) at 4°C for 1 h at 100 V. The membrane was blocked with Blocker Blotto (Thermo Scientific) at room temperature for 1 h. The membrane was incubated in anti-His rabbit antibody (1:2000) in Tris-buffered saline (0.1 M Tris, 0.9 % NaCl) added with 0.1 % Tween-20 and 1 % skim milk at 4°C on orbital shaker overnight. Membrane was washed three times in TBST, and alkaline phosphate goat antirabbit IgG antibody (1:5000) was added and incubated at room temperature for 1 h on orbital shaker. Unbound antibody was washed by three TBST washes and bands were visualized using an AP Conjugate Substrate Kit (BioRad).