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

Journal Article · Mon Jan 01 04:00:00 EST 2007 · BioTechniques

DOI:https://doi.org/10.2144/000112550· OSTI ID:932157

McDonald, W Hayes ^[1]; Huang, Ying ^[1]; Wu, Jun ^[1]; Liu, Yie ^[2]; Wang, Yisong ^[1]

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1. ORNL
2. National Institute on Aging, Baltimore

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{reg_sign}-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 x 10{sup 7} cells 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.

• Research Organization: Oak Ridge National Laboratory (ORNL); Bioprocessing Research Facility; Building Technologies Research and Integration Center; Mouse Genetics Research Facility
• Sponsoring Organization: ORNL LDRD Seed-Money; ORNL LDRD Director's R&D
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 932157
• Journal Information: BioTechniques, Journal Name: BioTechniques Journal Issue: 3 Vol. 43; ISSN BTNQDO; ISSN 0736-6205
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AFFINITY
CAPACITY
CLONING
DETECTION
MASS SPECTROSCOPY
PROTEINS
PURIFICATION
TELOMERES