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®-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to 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 approximately 16% from as little as 1-7 × 10 7cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)