SDS-PAGE for 35S Immunoprecipitation and Immunoprecipitation Western Blotting

This report discusses recent methods of sample preparation and gel electrophoresis for (35)S immunoprecipitation (IP) and IP western blotting. In both methods, IP is used to obtain purified proteins, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate the proteins on a gel. In (35)S IP, the proteins are radiolabeled and visualized on film by fluorography; in IP blotting, proteins are transferred onto nitrocellulose paper, and antibodies are used to detect specific proteins. A similar IP and SDS-PAGE method can be used for both procedures, but IP blotting has the potential advantages of improvement in sensitivity for low-abundance proteins and enhanced specificity for identification of proteins from a mixture. Some of the technical adaptations discussed here to facilitate IP blotting and avoid loss of beads or purified proteins may also be useful for (35)S IP.