Primary rabbit anti ha antibody

Transfected COS7 cells were homogenized in lysis buffer (150 mM NaCl, 50 mM Tris–Cl, pH 7.4, 5 mM EGTA, 5 mM EDTA, 1% Triton X-100, 25 lg/ml leupeptin, and 1 mM phenylmethylsulphonylfluoride) and maintained on ice for 30 min. After centrifugation at 10,000 g (4 °C) for 5 min, 100 μg of the supernatant was mixed 1:1 with SDS-PAGE sample buffer (50 mM Tris–Cl, pH 6.8, 50 mM DTT, 2% SDS, 10% glycerol, 5% βME). The proteins were separated in 10% SDS-PAGE gels and transferred electrophoretically to nitrocellulose membranes. After blocking with 5% skimmed dry milk in 1% TBS (pH 7.6)/0.1% Tween 20 for 1 h at room temperature, the blots were incubated with the primary rabbit anti-HA antibody (1:2000) (Abcam) for 1 hour. After washing, the first antibody was detected by a secondary sheep anti-rabbit IgG antibody (1:1000) conjugated with HorseRadish peroxidase (HRP, Amersham Biosciences) for 1 h at room temperature. Bands were visualized by the enhanced chemiluminiscence (ECL; Amersham Biosciences) and exposed to X-ray film. The blots were then stripped, blocked and incubated with the primary mouse anti-Myc antibody (1:1000) (Sigma) for 1 hour. Secondary goat anti-mouse IgG antibody conjugated with HRP 1:1000 (Amersham Biosciences) was incubated for 1 hour. Visualization was carried out as previously described^{57 (link)}.