Detection and analysis of interferon-alpha receptors on plasma membranes and in detergent extracts.
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.
Puvanakrishnan
R
R
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854-5635.
Langer
J A
JA
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003902:Detergents; D007370:Interferon Type I / Q000378:metabolism*; D008970:Molecular Weight; D011869:Radioligand Assay; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D017471:Receptors, Interferon; D012995:Solubility; D013154:Spleen / Q000276:immunology / Q000378:metabolism
D003902:Detergents; D007370:Interferon Type I; D011971:Receptors, Immunologic; D017471:Receptors, Interferon