Epr20579

After quantification of the EGFP binding by fluorescence, beads were boiled at 95 °C for 10 min in 15 μl 5× SDS loading dye (0.02% (w/v) bromophenol blue, 30% (v/v) glycerol, 10% (w/v) SDS, 250 mM Tris–HCl, 250 mM DTT, pH 6.8). Samples were separated by 12% Tris–glycine SDS-PAGE. After electrophoresis, the gel was blotted semi-dry on nitrocellulose membrane. As transfer buffer, 25 mM Tris, 100 mM glycine, 0.1% (w/v) SDS and 20% (v/v) methanol was used. The membrane was blocked for 1 h in 5% (w/v) nonfat milk powder containing Tris-buffered saline with TWEEN 20 (TBS-T, pH 7.4). Blocking was followed by three consecutive washing steps with TBS-T. Afterwards, the membrane was incubated with monoclonal primary anti-mCherry antibody (Abcam, EPR20579) derived from rabbit 1 : 1000 in blocking buffer at 8 °C overnight. Unbound primary antibody was removed in three washing steps with TBS-T. As the secondary antibody, anti-Rabbit IgG antibody (H  +  L) HRP conjugate produced in goat (Sigma-Aldrich) was used 1 : 10 000 in TBS-T incubated for 1 h. Three TBS-T washing steps followed the incubation with the secondary antibody. For chemiluminescent detection via the HRP, an ECL solution (Clarity Western ECL Substrate, Bio-Rad) was applied, and visualized by a Fusion FX imaging system (Vilber).