Western blot analysis of α-gal epitope expression on membrane glycoproteins

Twenty micrograms of processed or unprocessed membranes, including pig kidney membranes as a positive control, were subjected to 10% SDS-PAGE and then transferred electrophoretically onto a polyvinylidene difluoride membrane using a semi-dry electroblotting system.[18] The membrane was blocked overnight at 4°C with Blocking One (Nacalai Tesque, Japan), and then incubated with M86 anti-Gal mAb[18] (1:2 dilution) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 2 h at room temperature. After washing, the blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (1:1000 dilution) secondary Ab. The color reaction was developed using an ECL detection system (GE Healthcare, UK).