The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs
Book
·
OSTI ID:963907
- ORNL
Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter we describe a comprehensive methodology that utilizes our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we have demonstrated the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.
- Research Organization:
- Oak Ridge National Laboratory (ORNL); Building Technologies Research and Integration Center; Mouse Genetics Research Facility
- Sponsoring Organization:
- ORNL LDRD Seed-Money; ORNL LDRD Director's R&D; SC USDOE - Office of Science (SC)
- DOE Contract Number:
- AC05-00OR22725
- OSTI ID:
- 963907
- Country of Publication:
- United States
- Language:
- English
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