Epitope tagging of recombinant proteins

Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The fusion gene is cloned into an appropriate expression vector for the experimental cell type and host cells are transfected. The fusion protein can then be detected and/or purified using a monoclonal antibody specific for the epitope tag. This unit presents protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols in this unit employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG M2 affinity chromatography.