Demonstration of the transmembrane nature of the acetylcholine receptor by labeling with anti-receptor antibodies

Antibodies raised in rabbits to Triton-solubilized, purified acetylcholine receptor from Torpedo californica were used to immunospecifically label intact T. californica electroplaque membrane vesicles attached to cover slips and oriented with the extracellular face of the synaptic membrane facing outward. Hemocyanin conjugated to Protein A was then used as a marker, making the antibody binding visible at the electron microscope level. Parallel labeling experiments were performed on vesicles attached to cover slips and sheared by sonication, leaving their cytoplasmic faces fully exposed to the labeling solution. While differences in antibody populations among different rabbits were observed, antigenic determinants of the receptor were present on both faces of the membrane, with those on the extracellular side more numerous than those on the cytoplasmic side, demonstrating the transmembrane nature of the receptor molecule.