Detection and analysis of interferon-alpha receptors on plasma membranes and in detergent extracts

Abstract

The binding of interferons-alpha (IFN-alpha) to various cells is well characterized, but fewer studies have reported on the interaction of IFN-alpha with receptors on plasma membranes or in detergent-solubilized 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-solubilized 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.