Goat anti rabbit igg ap conjugate

4–12% Bis-Tris gels were loaded with 10 µl of precision plus protein kaleidoscope prestained protein standard from BioRad and 2 ug of protein in NuPAGE LDS 4X sample buffer (ThermoFisherScientific) per well. Proteins were run in duplicate. One gel was stained with Coomassie. The second gel was transferred using an iBlot 2 gel transfer device. The membrane was blocked for 1 h with 1X PBS/1% Casein blocker. Primary antibody (day 140 serum from gB protein vaccinated rabbit) was added to the blocking buffer and incubated overnight at 4°. Next day the membrane was washed 3X with 1XPBS with 0.1% Tween-20. Secondary antibody goat anti-rabbit IgG-AP conjugate (Sigma Cat. No.AP132A) at a 1:1000 dilution was added and incubated 1 h at room temperature. The membrane was washed 3X with 1XPBS with 0.1% Tween-20. Antibody binding was detected using western blue stabilized substrate for alkaline phosphatase. Gel and membrane were imaged with the molecular imager gel doc XR+ system with image lab software. All blots and gels derive from the same experiment and they were processed in parallel.

The contents of the proteins were tested by ID-ELISA according to the instructions for antibodies to determine the antiviral activity of VIGS. Leaves (0.2 g) were homogenized using a mortar and pestle and diluted 1: 3 in a PBS buffer. Crude extracts (100 μL) were added into ELISA plate wells and incubated at 37°C for 2 h. The plate was then washed with PBST buffer. TZSV NSs rabbit antibodies were diluted in a conjugation buffer, and afterward, 100 μL of goat anti-rabbit IgG-AP conjugate (Sigma, USA) was added to each well. The color-developing solution was dissolved in p-nitrophenyl phosphate disodium hexahydrate (Sigma-Aldrich) in substrate buffer to obtain a final concentration of 1 mg/mL. The absorbance was determined at 405 nm using the ELx 808 microplate ELISA reader (Bio-Tek, USA). Healthy leaves were used as negative controls, and TZSV-infected leaves were used as positive controls. The PBS buffer was used as a blank control.