Sypro ruby protein blot stain

Non‐denaturing 1–14% (v/v acrylamide) gradient gels (14 × 11 cm, 1.5 mm spacer) were manually cast (gel buffer 0.75 M aminocaproic acid, 50 mM BisTris, pH 7.0 and 0.1% CL47 detergent). 0.5 mg of Arabidopsis root membranes was solubilized in 0.5 ml of detergent buffer (CL47 and CL27, protein–detergent ratio of 1:10, with 1 mM EDTA/EGTA and protease inhibitors added) for 20 min on ice and cleared by ultracentrifugation (12 min at 100,000 g). The supernatant was supplemented with 10% sucrose and directly loaded onto the gel. After the run, gel lanes were excised, equilibrated for 2 × 10 min in 2× Laemmli buffer and loaded onto 10% SDS–PAGE gels for second dimension separation followed by Western blotting onto PVDF membranes. Western blot detection was carried out using mouse monoclonal anti‐PIN1 (Blilou et al, 2005) and HRP‐conjugated secondary ABs (Santa Cruz Biotechnology) and ECL Prime (Sigma‐Aldrich). Protein complexes visible in total protein stains of the respective Western blot membranes (SYPRO Ruby Protein Blot Stain, Bio‐Rad) were used as markers for the apparent molecular mass of complexes.

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).