Protein-blot analysis of receptor-ligand interactions.

Protein blotting was originally introduced as a solid-phase immunoassay (Gershoni & Palade, 1983; Towbin & Gordon, 1984). However, this technique can also be employed for the analysis of ligand-receptor interactions. In principle, a protein sample containing a receptor is resolved by gel electrophoresis and then blotted to an immobilizing matrix, such as a nitrocellulose membrane filter, as would be performed for immunoblotting. The blot is subsequently reacted with the corresponding ligand rather than a solution containing a particular immunoglobulin. The ligand-receptor complex is then detected, usually via autoradiography when the probe can be radioactively labelled. Whereas this corollary to blotting appears to be straightforward, there are a number of basic points that can jeopardize the chances of obtaining good results. Ligand overlay requires that binding capacity be maintained in the blotted counterpart. In immunodetection, the probe is a large, intact, native protein complex designed to recognize a distinct and small immobilized determinant. In the case of receptor detection, the probe might be extremely small and affinity and selectivity are expected of the protein which has undergone gel electrophoresis and blotting. These procedures are usually denaturative and so the dissociation and unfolding of the receptor components might readily destroy any quaternary and tertiary configurations required for the integrity of the ligand binding site. Another issue which should be considered is the detectability of the probe. Where iodinated ligands can be obtained, demonstration of a complex on the blot is easy. Many ligands, however, which are quite adequate for binding assays, are "Hor lJC-labelled and are thus less convenient for our purpose. Nonetheless, in spite of all the above apprehensions, there are quite a few systems for which protein blotting and ligand overlay have been very useful (for recent reviews see Gershoni, 19876, 1988). A case in point is the nicotinic acetylcholine receptor ( AChR).