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Nucleic Acids Research 2005 33(2):e23; doi:10.1093/nar/gni021
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Published online 1 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org.

Methods Online

Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins

Jinke Wang*, Tongxiang Li, Xiaoying Guo and Zuhong Lu

Chien-Shiung Wu Laboratory, Southeast University Nanjing 210096, China

*To whom correspondence should be addressed. Tel/Fax: +86 25 83619983; Email: wangjinke{at}seu.edu.cn

Received October 12, 2004. Revised December 2, 2004. Accepted January 14, 2005.

We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-{kappa}B, SP1 and p50, and the target protein of NF-{kappa}B in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

Correspondence may also be addressed to Zuhong Lu. Tel/Fax: +86 25 83619983; Email: zhlu{at}seu.edu.cn


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