LIGAND-BINDING ASSAYS WITH RECOMBINANT PROTEINS REFOLDED ON AN AFFINITY MATRIX

This paper describes a procedure for performing ligand binding assays with recombinant proteins ol protein fragments that can bind to an aff inity matrix in the presence or absence of a denaturing agent but whic h require the presence of the denaturing agent to remain in solution. The method involves coupling of a known amount of the protein in a den aturing medium to a known amount of the affinity matrix, replacing the denaturing agent with a physiological buffer and finally using the su spension of this protein-coupled matrix as the source of the recombina nt protein to be studied for its functional properties. A constant vol ume of this suspension is incubated with different concentrations of a radiolabeled ligand. Radioactivity bound to the protein-coupled affin ity matrix is determined after centrifugation and washing of the pelle t. Nonspecific binding is determined either by using the uncoupled aff inity matrix ol by the standard technique of measuring the binding in the presence of excess unlabeled ligand.