Anti gfp antibody ab6556

Strains with recombinant plasmids were cultured to the exponential phase and 1-mL bacterial suspension was collected by centrifugation at 12,000 × g for 1 min at room temperature. The pellet was suspended in 40 μL ddH₂O and 40 μL 2× SDS sample buffer (25 mM Tris pH 6.8, 0.8% SDS, 4% glycerol, 2% 2-mercaptoethanol, and 0.005% bromophenol blue). Samples were boiled for 15 min and separated by 12% SDS-PAGE. Proteins separated by SDS-PAGE were electrotransferred to a PVDF membrane under a constant 15V. The PVDF membrane was blocked in TBST (0.3% Tris, 0.88% Nacl, 0.02% KCl, 0.05% Tween20, pH = 7.4) containing 5% dried skim milk for 1 h at 37 °C. The membrane was incubated with a rabbit anti-GFP antibody (anti-GFP antibody ab6556, Abcam, UK) diluted 1:1000 in TBST for 1 h at room temperature. The membrane was then washed in TBST and incubated with a goat anti-rabbit IgG antibody diluted 1:1000 in TBST for 1 h at room temperature. Immunoreactivity was visualized using an imaging apparatus (Tanon 5200, China) with enhanced chemiluminescence solution (SuperSignal West Pico Stable Peroxide Solution, PC 199723; SuperSignal West Pico Luminol Enhance Solution, PC 199939, Thermo, USA).