Ap coupled goat anti rabbit igg

Total protein extraction and western blotting were performed as described (Zhuo et al., 2014 (link)). Briefly, plant total proteins were extracted by grinding 0.1 g N. benthamiana leaves in 300 μl of 2× SDS-PAGE buffer with subsequent heating at 100°C for 10 min. Protein samples were separated by electrophoresis in 12.5% SDS-PAGE and transferred onto Nitrocellulose Membrane (GE Healthcare). Membranes were blocked in 1× TBST buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) with 5% non-fat milk at 37°C for 1 h, washed three times (10 min each time) with 1× TBST buffer, followed by AP-coupled goat anti-rabbit IgG (Sigma) and NBT (0.33 mg/ml)/BCIP (0.165 mg/ml) staining. BrYV CP, MP, CP-RTD, and PEMV 2 MP were expressed and purified from Escherichia coli, and the purified proteins were used for generation of the polyclonal antibodies in rabbits at Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The antisera raised against BrYV CP, MP, CP-RTD, and PEMV 2 MP were used to detect the accumulation of BrYV and PEMV 2 in N. benthamiana. Antisera raised against Flag (Sigma), MBP (GenScript), RFP (GenScript), and GFP (GenScript) were used to detect the expression of P3a fusion proteins.

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).