title: Filter-binding assay procedure for thyroid hormone receptors.
abstract: An assay procedure for thyroid hormone receptor activity which used nitrocellulose membrane filters was developed. Receptor proteins, extracted from washed rat liver nuclei with a 0.4 M NaCl solution, were incubated with 125I-labeled thyroid hormone (T3), and filtered on the cellulose ester membranes under suction at 2 degrees C. The filters were subsequently washed with cold buffer and counted for 125I radioactivity. The method allowed an accurate estimation of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentrations. The usefulness of this filter-binding method became evident when it was compared with the conventional procedure that employs Sephadex G-25 columns. For practical application to routine assays, various filtration conditions were examined, and a standard procedure was established. Using this technique, the isolated receptors were determined to possess an apparent Kd of 1.38 X 10(-10) M and a pH optimum of T3 binding at 8.2-8.4.
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D005374:Filtration; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D008297:Male; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000032:analysis*; D011988:Receptors, Thyroid Hormone; D012995:Solubility
chemical_list: D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone