Hrp conjugated sheep anti mouse 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).

Peptides were coated on a polyvinyl chloride (PVC) microtiter plate at 10 µg/mL in buffer (0.2 M NaHCO₃ buffer, pH 9.6) overnight at 4 °C and non-specific binding was blocked with 2% bovine serum albumin (BSA) for 1 h at RT. After washing (0.05% Tween 20/phosphate buffered saline (PBS)), serial dilutions of the anti-MUC1 antibody, BC2 recognizing SAPDTRPAP (MUC1K^b) or 1.3.14 recognizing STAPPAHGV (MUC1A2) were added and incubated for a further 1 h at RT. Plates were then washed and bound antibody was detected using HRP-conjugated sheep anti-mouse antibody (Amersham, UK). Responses were detected with TMB substrate solution (Invitrogen, Carlsbad, CA, USA) and stopped with 1M HCl. Absorbance was read at 450 nm.