Horseradish peroxidase hrp conjugated goat anti rabbit antibody

A lysis buffer (Beyotime Institute of Biotechnology, Nantong, China) was used to prepare total protein extracts from cell lines as well as tumor tissues. Then, protein concentrations were evaluated by a BCA kit (Beyotime Institute of Biotechnology, Nantong, China). An equal protein amount (40 μg per lane) was separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 12% acrylamide gels and transferred onto polyvinylidine difluoride (PVDF) membranes (Millipore Corporation, Billerica, USA) which were blocked in 5% fat-free milk followed by overnight incubation at 4°C with the rabbit anti-NUF2 primary antibody (1 : 5000 dilution; Sangon Biotech, Shanghai, China). The secondary antibody was horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody (1 : 2000; Beyotime Institute of Biotechnology). After stripping, the membrane was reprobed overnight at 4°C with antibody against GAPDH (1 : 2000; Beyotime Institute of Biotechnology), followed by incubation with secondary antibodies as above at room temperature (RT) for 2 h. An enhanced chemiluminescence system (ECL; Beyotime Institute of Biotechnology) was used for band visualization. The band intensities were quantified by densitometry.