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Title: The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs

Abstract

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.

Authors:
 [1];  [1]
  1. ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Building Technologies Research and Integration Center (BTRIC); Mouse Genetics Research Facility
Sponsoring Org.:
USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE Office of Science (SC)
OSTI Identifier:
963907
DOE Contract Number:  
DE-AC05-00OR22725
Resource Type:
Book
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AFFINITY; DETECTION; MASS SPECTROSCOPY; MONITORING; PROTEINS; PURIFICATION

Citation Formats

Giannone, Richard J, Liu, Yie, and Wang, Yisong. The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs. United States: N. p., 2009. Web.
Giannone, Richard J, Liu, Yie, & Wang, Yisong. The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs. United States.
Giannone, Richard J, Liu, Yie, and Wang, Yisong. 2009. "The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs". United States.
@article{osti_963907,
title = {The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs},
author = {Giannone, Richard J and Liu, Yie and Wang, Yisong},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/963907}, journal = {},
number = ,
volume = ,
place = {United States},
year = {Thu Jan 01 00:00:00 EST 2009},
month = {Thu Jan 01 00:00:00 EST 2009}
}

Book:
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