Tris pH 7.4. Transfer lysate to a 1.5ml microcentrifuge tube and boil for an additional 5 minutes. Scrape the cells from the dish and pass several times through a 26 gauge needle to disperse any large aggregates.
Repeat wash twice.
Load the supernatant onto an SDS-PAGE gel and electrophorese. When radiolabeled cells are used for immunoprecipitation, higher backgrounds may result. In this case, more stringent washing pro- cedures may be required. Cold Spring Harbor Laboratory, 1988.