Visualization of the insulin receptor by immunoblotting.
Insulin receptors from rat hepatoma cells (Fao) and human placenta were partially purified by detergent solubilization and lectin purification. The insulin receptor preparations were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing or nonreducing conditions. The proteins were transferred to nitrocellulose paper and the electrophoretic blots were treated with human anti-receptor autoantibodies, rabbit antibody to purified insulin receptor, or a monoclonal antibody to human insulin receptor. The nitrocellulose paper was then treated with 125I protein A or 125I second antibody followed by autoradiography. The rabbit polyclonal antiserum and one of the human autoantibodies recognized both the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits after transfer from a SDS gel to nitrocellulose paper. On transfers from nonreduced gels, several high-molecular species were labeled ranging from Mr = 200,000 to Mr = 330,000. Similar high-molecular bands of the receptor were seen if highly purified human placental receptor, as well as partially purified receptor from rat or human origin, were used. As little as 0.1-0.5 microgram of pure receptor could be detected by this technique. Treatment of the receptor with neuraminidase (50 mU/ml) before gel electrophoresis resulted in a 50% increase in intensity of intact receptor and about a 70% increase in the labeling of the alpha-subunit of the receptor, but no change in labeling of the beta-subunit. The monoclonal antibody used, as well as two other human autoantibodies, did not recognize the receptor after transfer to nitrocellulose paper.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001345:Autoradiography; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D011972:Receptor, Insulin