Western Blot Analysis of Piscine Reovirus Proteins

Purified PRV-3 virus particles or blood pellets pooled from three cohabitant fish were mixed with XT buffer and XT reducing agent (Bio-rad), heated for 5 min at 95 °C and loaded onto a 4–12% criterion XT bis–tris gel. Separated proteins were transferred onto a 0.2 µm PVDF membrane using Trans Blot Turbo Transfer system (Bio-rad) and incubated overnight at 4 °C with antiserum against PRV-1 proteins; anti-σ1 (1:1000) [19 (link)], anti-σ3 (1:500) [1 (link)], anti-µ1C (1:500) [19 (link)] and anti-actin (1:500) (Sigma). After washing 4 × 15 min the membranes were incubated with the secondary antibody horse radish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham, GE Healthcare, Buchinghamshire, UK) (1:20 000). The membranes were washed 4 × 15 min and stained with the Clarity Western ECL Substrate kit (Bio-rad). MagicMark was used as molecular weight ladder (XP Western Protein Standard, Invitrogen). Images were acquired using ChemiDoc XRS+ system and Image one software (Bio-rad).