Rabbit anti gfp antibody

Total proteins were harvested from leaf samples as described previously by Sun et al. (2018) using 2× SDS loading buffer (100 mm Tris (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue) containing 10% β-mercaptoethanol. Proteins were isolated on 12.5% SDS polyacrylamide gels and then transferred onto polyvinylidene fluoride membranes (Ge Healthcare, Buckinghamshire, UK). The membranes were blotted with a rabbit anti-flag antibody (1:1000; Sigma–Aldrich, St. Louis, MO, USA), mouse anti-flag antibody (1:5000; Sigma–Aldrich), rabbit anti-GFP antibody (1:3000; Genscript, Nanjing, China) or rabbit anti-PLRV MP antibody (1:5000) [53 (link)]. Immunoreactive proteins were successively visualized by blotting with goat anti-rabbit AP antibody (1:10,000; Sigma–Aldrich), goat anti-mouse AP antibody (1:10,000; Sigma–Aldrich), goat anti-rabbit HRP antibody (1:3000; Sigma–Aldrich) or goat anti-mouse HRP antibody (1:3000; Bio-Rad, Hercules, CA, USA) followed by visualization using nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate (Sigma–Aldrich) or a chemiluminescence detection kit (GE Healthcare).

Protein extraction and western blot analysis were performed as described previously [30 (link)]. Briefly, total proteins were extracted from 0.1-g leaf samples by grinding into fine powder in liquid nitrogen, followed by adding 300 µL of 2 × SDS buffer (100 mM Tris (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, and 0.2% (w/v) bromophenol blue) with subsequent heating at 100 °C for 10 min. Proteins were separated in 12.5% SDS polyacrylamide gel by electrophoresis and transferred to nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) using a mini trans-blot electrophoretic transfer cell (Bio-Rad).
Western blot analysis was performed using the rabbit anti-flag antibody (Sigma-Aldrich, St Louis, MO, USA) diluted at 1:1000, or mouse anti-flag antibody (Sigma-Aldrich) diluted at 1:5000, or rabbit anti-GFP antibody (GenScript, Nanjing, China) diluted at 1:3000, or rabbit anti-PLRV movement protein (MP) antibody [45 (link)] diluted at 1:5000, as a primary antibody. Incubation was then performed with goat anti-rabbit IgG (Sigma-Aldrich) diluted at 1:3000, or goat anti-mouse HRP (Bio-Rad) diluted at 1:3000, as a secondary antibody. Finally, the membranes were detected using the nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate (Sigma-Aldrich), or with a superior chemiluminescence detection kit (GE Healthcare) according to the manufacturer’s instructions.