Coomassie brilliant blue stain

Eluted fractions following analytical-SEC were further examined by SDS–PAGE (12% gels) (Bio-rad, California, U.S.A.) and bands were visualised using a Silver staining kit (Invitrogen, California, U.S.A.) for analytical-SEC fraction analysis and Coomassie Brilliant Blue stain (ThermoFisher, Massachusetts, U.S.A.) for band density analysis. Both techniques were performed according to the manufacturer's instructions. Band densities were calculated using GelAnalyzer 9.1 (http://www.gelanalyzer.com/).

The supernatants (100 µL) were concentrated with a centrifugal filter (Microcon YM-30; Merck, Darmstadt, Germany) by centrifuge ×14,000 g at 25℃ for 30 minutes, then solubilized in 2× sample buffer and treated at100℃ for 5 minutes before use. The E2 CSFV recombinant protein (10 µL) was reduced with 2.5% β-mercaptoethanol. Proteins were either stained with Coomassie Brilliant Blue Stain (Thermo Scientific) or transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen) for western blotting using Mini Trans-Blot cell system (BioRad, Hercules, CA, USA). The transferred PVDF membrane was blocked with 5% nonfat milk (Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST). The membrane was incubated with mAb anti-CSFV antibody (The National Institute of Animal Health, Tsukuba, Japan) for 1 hour at 25℃, washed 3 times with PBST. Then, the membrane was incubated with POD-anti-mIgG (H+L) (GE Healthcare, Chalfont St. Giles, UK) for 1 hour at 25℃ and washed 3 times with PBST. Detection of immunoreactive bands was performed using Western Lightning Plus-ECL substrate (Thermo Scientific) as per the supplier's instructions.