A key component in determining the functional role of any protein is the elucidation of its binding partners using protein-protein interaction (PPI) data. Here we examine the use of tandem affinity purification (TAP) tagging to study RNA/DNA helicase PPIs in Escherichia coli. The tag, which consists of a calmodulin-binding region, a TEV protease recognition sequence, and an IgG-binding domain, is introduced into E. coli using a lambdared recombination system. This method prevents the overproduction of the target protein, which could generate false interactions. The interacting proteins are then affinity purified using double affinity purification steps and are separated by SDS-PAGE followed by mass spectrometry identification. Each protein identified would represent a physical interaction in the cell. These interactions may potentially be mediated by an RNA/DNA template, for which the helicase would likely be needed to disrupt the secondary structures.