Immunoprecipitation of GFP-Tagged Proteins

Total proteins were extracted from one gram of co-inoculated leaves of N. benthamiana by using 2 mL of ice-cold immunoprecipitation buffer (10% [v/v] glycerol, 25 mM Tris-HCI, pH 7.5, 150 mM NaCl, 10 mM DTT, 1 mM EDTA, 1 × Protease Inhibitor Cocktail, For Plant Cell (Sangon Biotech), and 0.15% [v/v] Nonidet P-40). Protein extracts were incubated with GFP-Trap beads (ChromoTek) for 1h at 4°C. The beads were collected and washed with the buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Nonidet P40, 0.5 mM EDTA). Total protein extracts prior to (Input) and after immunoprecipitation (IP) were analyzed by immunoblotting using anti-GFP and anti-Myc polyclonal antibodies (Abcam), essentially as previously described [63 (link)].