Rabbit polyclonal anti gfp antibody

Western blotting was carried out as described previously (Asakawa et al. 2014) with slight modifications. S. pombe cells expressing Lem2-GFP (cYK183-3A) were cultured in the YES or EMMG medium to log phase for 24 h. After NaOH/ trichloroacetic acid precipitation, the pellets were resuspended in sample buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel. After SDS-PAGE, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by wet transfer. The membrane was blocked in 5% skim milk and incubated with a rabbit polyclonal anti-GFP antibody (2.5 lg/mL; Rockland Immunochemicals Inc.) for overnight at 4 °C. The membrane was washed with PBS containing 0.05% Tween â -20 three times and then incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1 : 5000; GE Healthcare) for 2 h at room temperature. Bands were detected by chemiluminescence using a ChemiDoc TM Roles of Lem2 in genome stability MP imaging system (Bio-Rad). For the loading control, actin was detected by a mouse monoclonal anti-b-actin antibody (1.5 lg/mL; Abcam) and an HRP-conjugated sheep antimouse IgG.