Hrp conjugated anti mouse rabbit antibody

Total protein was extracted from the chicken oviduct as a positive control and from chicken leg tissue and embryonic fibroblast cells (DF-1) as negative controls. Total protein was also extracted from the cOECs. RIPA lysis buffer (Thermo Scientific, Waltham, MA) containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA) was used for protein extraction. The protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA), and 15 μg of protein extract was electrophoresed using an SDS-PAGE 4 to 12% gel system (Invitrogen, Carlsbad, CA). Separated proteins were transferred to a polyvinylidene fluoride membrane (PVDF; Invitrogen, Carlsbad, CA). The membrane was blocked with 5% skim milk in PBS (Thermo Scientific, Waltham, MA) for 1 h and incubated with a rabbit polyclonal anti-ovalbumin antibody (1:1,000; 1 mg/mL; Abcam, Cambridge, UK) and rabbit monoclonal anti-vinculin antibody (1:1,000; 0.054 mg/mL; Abcam, Cambridge, UK) overnight at 4°C. The membrane was washed and incubated with a mouse anti-rabbit HRP-conjugated antibody (1:2,000; 0.4 mg/mL; Santa Cruz, Dallas, TX) for 30 min at room temperature. Amersham ECL prime (GE Healthcare, Buckinghamshire, UK) substrate was used to visualize the target bands, and the bands were analyzed using EZ-Capture II (Atto, Tokyo, Japan).