Immunoblot detection of antigens in immunoprecipitates

摘要

Co-immunoprecipitation of antigens from cell or tissue extracts is used in a wide variety of experimental systems to corroborate the existence of authentic protein-protein interactions in vivo. Typically, stable protein-protein associations are detected by immunoprecipitation with antibodies specific for one protein, followed by immunoblot analysis with antibodies against potential interacting proteins. However, in many cases, the only immunoreagents available for blotting are antibodies that have been generated in the same animal species as the antibodies used for immunoprecipitation. This can give rise to extremely high levels of background staining on blots because secondary antibody conjugates bind directly to the electrophoresed immunoglobulins in the immunoprecipitate. We have overcome this problem by probing immunoblots of immunoprecipitates with biotinylated antibodies, followed by an avidin-peroxidase conjugate.