Detection and Analysis of Interferon-α Receptors on Plasma Membranes and in Detergent Extracts

Publisher Website
Google Scholar
Abstract
The binding of interferons-.alpha. (IFN-.alpha.) to various cells is well characterized, but fewer studies have reported on the interaction of INF-.alpha. with receptors on plasma membranes or in detergent-solublized form. We describe a simple, sensitive, and semiquantitative assay procedure to detect the presence of IFN-.alpha. receptors on bovine spleen plasma membrane preparations or in detergent-solublized extracts. The procedure involves spotting the sample on hydrophobic polyvinylidene difluoride (PVDF; Immobilon P) membranes, blocking the filter with milk, and binding radiolabeled IFN-.alpha.A to the membrane filter, with detection by either autoradiography or scintillation counting. This assay procedure has been applied for the identification of IFN-.alpha. receptors in crude and affinity-purified fractions. The partially purified IFN-.alpha. receptors have been further characterized by SDS-polyacrylamide gel electrophoresis (PAGE). The separated IFN-.alpha. receptor protein on the SDS-PAGE gel has been electrophoretically transferred to Immobilon membrane and visualized by ligand blotting. This provides an estimate of 95-110 kD for the apparent molecular weight and a tool for further studies of the receptor protein.