Colorimetric ap substrate reagent kit

Mutant rAg, WT rAg as a positive control, and diluent as a negative control were separated by SDS-PAGE using 4–20% Tris-HCl Criterion gel (Bio-Rad, CA, USA) under reducing conditions at 1.0 μg load, and then transferred onto nitrocellulose membranes using a Trans-Blot Turbo system according to the manufacturer’s protocol (Bio-Rad). After protein transfer, one membrane was stained with SYPRO™ Ruby protein blot stain according to the manufacturer’s protocol (Bio-Rad). The remaining nitrocellulose membranes were treated with T20 Blocking buffer (Thermo Fisher Scientific) for 60 min, followed by three 5-min washes in 1× TRIS buffered saline (TBS). The membranes were then incubated with 1.0 μg/ml of anti-nucleocapsid antibodies (Ab1, Ab2, Ab3 or Ab4; see Supplementary Table 2) prepared in antibody buffer (1× TBS, 1% BSA, 0.05% Tween^® 20) for 16–18 h at room temperature, followed by three 5-min washes (1× TBS). The membranes were then incubated with alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (H&L)-AP or goat anti-human IgG (H&L)-AP antibody (Bio-Rad) at 1:2000 dilution prepared in antibody buffer (1× TBS, 1% BSA, 0.05% Tween 20) for 2 h at room temperature, followed by three 5-min washes (1× TBS). The blots were developed with colorimetric AP substrate reagent kit according to the manufacturer’s protocols (Bio-Rad).