Purification of TAP-tagged proteins by two-step pull down from DT40 cells.

Abstract

For proteomic analysis, protein purification from cell extracts is an important step. Since production of high quality antibody is time consuming and not guaranteed to be successful, expression of epitope-tag conjugated protein of interest followed by immunoprecipitation using anti-epitope-tag antibody is a common method for protein purification. Here we describe use of an epitope-tag called TAP (tandem affinity purification) in DT40, which consists of Protein A IgG-binding motif and calmodulin binding motif separated by TEV cleavage site. Tandem purification using two different epitopes should eliminate non-specific binding and help identifying physiological protein-protein associations.