Rabbit anti human parp polyclonal antibody

Cells were lysed with raidoimmunoprecipitation assay (RIPA) reagent (Beyotime, ShangHai, China) in the presence of protease inhibitors (Sigma, USA) and total protein quantified with the bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). A total of 20 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Expression of cleaved caspase 8 was detected using mouse anti-human caspase 8 monoclonal antibody (Cell Signaling Technology, Beverly, MA) and HRP-conjugated sheep anti-mouse antibody (Amersham Bioscience). Expression of cleaved PARP was detected using rabbit anti-human PARP polyclonal antibody (Cell Signaling Technology) and horseradish peroxidase (HRP)-conjugated sheep anti-rabbit antibody (Amersham Bioscience). Expression of GAPDH was detected using rabbit anti-human GAPDH polyclonal antibody (Amersham Bioscience) and HRP-conjugated sheep anti-rabbit antibody (Amersham Bioscience). Protein expression was visualized using ECL Plus reagent (Amersham Bioscience) and a Chemilumino analyzer GelDoc XR system (Biorad, USA).

The proteins were separated by molecular weight using 10% SDS-PAGE gels (Sigma-Aldrich) and were transferred onto a nitrocellulose membrane (Advantec MFS, Inc., Dublin, CA, USA). The membrane was subsequently blocked in 5% non-fat milk at room temperature for 60 min.
The membranes were then incubated with the primary antibody (rabbit anti-human PARP polyclonal antibody; #9664; Cell Signaling Technology, Inc., MA, USA) diluted in 5% non-fat milk (Fuzhou Maixin Biotechnology Development Co., Ltd.) overnight at 4°C. Following washing in Tris-buffered saline containing Tween-20 (TBST; Shanghai Sangon, Biological Engineering Co., Ltd.) three times for 10 min on a horizontal shaker (HZ-9611K; Hualida Experiment Equipment Company, Nanjing, China), the membranes were incubated with the secondary antibody (horseradish-peroxidase-labeled goat anti-rabbit IgG; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) diluted in 5% non-fat milk at room temperature for 60 min and were washed with TBST and stained with DAB coloration liquid (Sigma-Aldrich), followed by developing with the ECL system (GE Healthcare Life Sciences, Livingston, NJ, USA) according to the manufacturer’s instructions.