Analyzing MUC1 Expression on Cell Surfaces

To investigate MUC1 expression on the surface of MUC1+B16 cell, MCF7 and MUC1+B16 cell surface protein were isolated using Cell Surface Protein Isolation Kit(Pierce) according to the standard manual. The isolated cell surface protein samples were analyzed by western blotting. Samples were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated overnight at 4°C with anti-MUC1 antibody(abcam, ab22711), followed by FITC-flourecent goat anti mouse IgG and finally visualized using Odyssey fluorescence imaging system(LICOR, US). To further investigate the binding profiles of anti-serum from the CTB-MUC1-alum-CpG group with native human MUC1, surface plasmon resonance analysis was conducted as previously described [40 (link)]. cells were blocked in phosphate-buffered saline (PBS) supplemented with 5% BSA and then incubated at 37°C with anti-serum from the CTB-MUC1-alum-CpG group. (1:100) for 1 h. The cells were then washed three times with PBS (pH 7.4) and then incubated at 37°C with PE-labeled rat anti-mouse IgG1 for another 1 h and fixed before labeling the nuclei with Hoechst 33258. Next, the cells were observed with a flouresent microscope (Zeiss).