Filter-binding assay procedure for thyroid hormone receptors

✍ Scribed by Akira Inoue; Junichi Yamakawa; Munehiko Yukioka; Seiji Morisawa

• Book ID: 102984785
• Publisher: Elsevier Science
• Year: 1983
• Tongue: English
• Weight: 666 KB
• Volume: 134
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(83)90280-4

✦ Synopsis

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.