Western blot analysis of the α1A subunit of voltage-gated calcium channels in human erythrocyte ghosts from 3 different donors.
Red cell membrane proteins were separated electrophoretically at high protein loads (80 μg). After electrophoretic transfer to nitrocellulose paper, blots were stained with antibodies directed against residues 865 to 881 of the α1A subunit of the rat brain voltage-gated calcium channel (lanes 1A, 2A, 3A). Because several nonspecific bands were also visualized, competition of Cav2.1 antibody staining using its specific peptide was also performed. For this purpose, 10 μg of the Cav2.1 antibody was preincubated with 10 μg of antibody-specific peptide for 1 hour at 22°C and then further incubated with the blot for 2 hours at 22°C (lanes 1B, 2B, 3B). Polypeptides with Mr of approximately 190 000 and 220 000 are characteristic of the major splicing variants of the α1A subunit of Cav2.1 in the brain. An SDS-PAGE Coomassie blue–stained gel of RBC membranes (left lane; “RBC Ghost”) serves as an approximate molecular weight marker.
