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Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins
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 f...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2005
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548379/ https://www.ncbi.nlm.nih.gov/pubmed/15687381 http://dx.doi.org/10.1093/nar/gni021 |
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author | Wang, Jinke Li, Tongxiang Guo, Xiaoying Lu, Zuhong |
author_facet | Wang, Jinke Li, Tongxiang Guo, Xiaoying Lu, Zuhong |
author_sort | Wang, Jinke |
collection | PubMed |
description | 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-κB, SP1 and p50, and the target protein of NF-κB 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. |
format | Text |
id | pubmed-548379 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2005 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-5483792005-02-10 Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins Wang, Jinke Li, Tongxiang Guo, Xiaoying Lu, Zuhong Nucleic Acids Res Methods Online 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-κB, SP1 and p50, and the target protein of NF-κB 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. Oxford University Press 2005 2005-02-01 /pmc/articles/PMC548379/ /pubmed/15687381 http://dx.doi.org/10.1093/nar/gni021 Text en © The Author 2005. Published by Oxford University Press. All rights reserved |
spellingShingle | Methods Online Wang, Jinke Li, Tongxiang Guo, Xiaoying Lu, Zuhong Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins |
title | Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins |
title_full | Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins |
title_fullStr | Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins |
title_full_unstemmed | Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins |
title_short | Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins |
title_sort | exonuclease iii protection assay with fret probe for detecting dna-binding proteins |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548379/ https://www.ncbi.nlm.nih.gov/pubmed/15687381 http://dx.doi.org/10.1093/nar/gni021 |
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