Dual-tagging system for the affinity purification of mammalian protein complexes

Although affinity purification coupled with mass spectrometry (MS)provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway (R)-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and mrious combinations ofa ini facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approxiniately 16%from as little as 1-7 x 10(7) cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new toolfor expanding the capacity, to explore mammalian proteomic networks.