Combining Enhanced Metabolic Labeling with Immunoblotting to Detect Interactions of Endogenous Cellular Proteins

Abstract

Metabolic labeling, immunoblotting and two-dimensional isoelectric focusing/SDS-PAGE are powerful techniques for characterizing endogenously expressed cellular proteins and their interactions. We achieved improved resolution and sensitivity for the detection of metabolically labeled proteins separated on two-dimensional gels by electroblotting the proteins onto polyvinylidene difluoride or nitrocellulose membranes and detecting the 35S signal on a bio-image analyzer. We obtained independent detection of specific proteins from the same blot by subsequent rehydration of the membrane and immunoblot analysis. The combination of these enhanced detection techniques with immunoprecipitation and two-dimensional electrophoresis on precast minigels provides a simple, sensitive method for detecting interactions between endogenous proteins in the cell.