Simultaneous measurements of rates of protein de novo synthesis and its steady-state levels by immunoblotting

Abstract

Immunoblotting was used for simultaneous measurements of rates of de novo synthesis and steady-state levels of a model protein—cytochrome P450r. This is the major phenobarbital-inducible P450 form in cultured chick-embryo hepatocytes (L. Oron, and S. Bar-Nun, (1984) Biochem. Biophys. Res. Commun. 125, 1096–1102) . Cultured hepatocytes were metabolically labeled with [ 35S]methionine, and cellular proteins were resolved by gel electrophoresis and then quantitatively electroblotted onto nitrocellulose. The blot was directly exposed to autoradiography and the rates of de novo synthesis were estimated from the 35S label of P450r. The same blot was then incubated with specific anti-P450r antibodies, followed by 125I-labeled secondary antibodies, and the blot was reexposed to autoradiography through white paper. The paper masked the 35S-labeled proteins and the steady-state levels were estimated from the 125I-immunodecorated P450r. By this simple method, “true” inducers of cytochrome P450r, such as phenobarbital, can be defined, whereas “false” inducers, which primarily affect the degradation of P450r, can be detected.