Application of the miniature ultracentrifuge in receptor-binding assays

A new binding assay for membrane receptor systems has been developed employing an air-driven ultracentrifuge (Beckman Airfuge). The main advantages of this method for measurement of radioligand binding in aqueous medium are (i) the rapidity (30 s) in separating the bound from the unbound fraction, (ii) the small volume (100 μl) of assay medium which permits a relatively small excess of ligand over receptor to be employed, and (iii) the simplicity of manipulations which allows a high degree of replication. The variation in a triplicate set of assays is usually less than 0.5%. By virtue of maintaining equilibrium throughout the assay the present method is especially useful for ligands exhibiting rapid reversibility in binding. Binding of [3H]ouabain to several membrane (Na+, K+)-ATPases and binding of [3H]etorphine to the oplate receptor from brain membranes are discussed here. Also the inhibition of [3H]ouabain binding by Tris is discussed.