Western lightning plus ecl substrate

Western blotting was performed as described previously (Truong et al., 2017 (link); Truong et al., 2018a ; Truong et al., 2018b (link)). Recombinant proteins (100 ng/µL) were subjected to SDS-PAGE, and the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen) using a mini Trans-Blot System (Bio-Rad, CA) according to the manufacturer's protocol. The transferred PVDF membrane was blocked with 5% non-fat milk Sigma-Aldrich (St. Louis, MO) dissolved in PBS containing 0.05% Tween 20 (PBST). The membrane was incubated with the primary antibody overnight at 4°C, washed thrice with PBST, and further incubated with HRP-conjugated secondary antibodies for 1 h at room temperature (25°C). Immunoreactive bands were detected using Western Lightning Plus-ECL substrate (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions.

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.