Specific primary anti gfp polyclonal antibodies

All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with the specific primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3 × 10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10000 with milk buffer). Specific signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scientific™) according to the manufacturer’s manual. Finally, the membranes were exposed to Kodak BioLight film.