5 bromo 4 chloro 3 indolylphosphate nitroblue tetrazolium bcip nbt solution

The levels of the TSWV proteins N and NSm were analyzed by western blotting. Total protein was extracted from tobacco leaves (0.2 g, fresh weight) using TriPure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Equal sample volumes (20 μL) were loaded on a 12.5% polyacrylamide gel, and proteins were separated by electrophoresis at 100 V for 90 min. After being transferred to a PVDF membrane, the N and NSm proteins were detected using a primary antibody (1: 4,000) and were subsequently probed with AP-coupled goat anti-rabbit IgG (1:8,000; Sigma, Santa clara, USA). The signals on the membrane were visualized using a ready-for-use 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) solution (Sangon Biotech, Shanghai, China).

Protein concentrations were determined using the Bio-Rad protein assay kit. Protein samples were treated with 3× SDS buffer. After boiling for 7 min, the samples were separated by electrophoresis in 10% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The antigens on the membrane were incubated with a monoclonal antibody against TSWV N (1:10,000 dilution) (29 ) and subsequently probed with AP-coupled goat anti-mouse IgG (1:10,000 dilution; Sigma). The signals on the membrane were visualized with ready-for-use 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) solution (Sangon Biotech, Shanghai, China).