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Title: FRET-based protein-DNA binding assay for detection of active NF-kappa B

Abstract

A novel method to detect the active form of NF-{kappa}B, a transcription factor regulating a battery of inflammatory genes and playing a fundamental role in the development of numerous pathological states, has been developed. In the present work, we used fluorescence resonance energy transfer (FRET) to study DNA-protein binding interaction taking place between double-strand (ds) DNA immobilized in a glass capillary wall and p50 proteins. For this purpose, we developed a regenerable FRET-based system comprising of a single-strand (ss) DNA with auto-complementary sequence that is end-labeled with Cy5 dye and is highly specific for p50 proteins. The proteins were labeled with a Black Hole Quencher (BHQ-3) to be used as FRET pair. The interaction of p50/p50 homodimer active form with its DNA binding site was demonstrated by both electrophoretic mobility shift assays and FRET studies. These preliminary results demonstrated the feasibility of the FRET-based DNA technique to detect the active form of NF-{kappa}B protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable.

Authors:
 [1];  [1];  [1];  [1]
  1. ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
989529
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Sensors and Actuators B: Chemical; Journal Volume: 113; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSICS; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; BLACK HOLES; DETECTION; DNA; DYES; EFFICIENCY; ENERGY TRANSFER; FLUORESCENCE; GENES; GLASS; PROTEINS; RESONANCE; TRANSCRIPTION FACTORS; COMPLEX; REL

Citation Formats

Giannetti, Ambra, Baldini, Francesco, Wabuyele, Musundi B, and Vo Dinh, Tuan. FRET-based protein-DNA binding assay for detection of active NF-kappa B. United States: N. p., 2006. Web. doi:10.1016/j.snb.2005.07.014.
Giannetti, Ambra, Baldini, Francesco, Wabuyele, Musundi B, & Vo Dinh, Tuan. FRET-based protein-DNA binding assay for detection of active NF-kappa B. United States. doi:10.1016/j.snb.2005.07.014.
Giannetti, Ambra, Baldini, Francesco, Wabuyele, Musundi B, and Vo Dinh, Tuan. Sun . "FRET-based protein-DNA binding assay for detection of active NF-kappa B". United States. doi:10.1016/j.snb.2005.07.014.
@article{osti_989529,
title = {FRET-based protein-DNA binding assay for detection of active NF-kappa B},
author = {Giannetti, Ambra and Baldini, Francesco and Wabuyele, Musundi B and Vo Dinh, Tuan},
abstractNote = {A novel method to detect the active form of NF-{kappa}B, a transcription factor regulating a battery of inflammatory genes and playing a fundamental role in the development of numerous pathological states, has been developed. In the present work, we used fluorescence resonance energy transfer (FRET) to study DNA-protein binding interaction taking place between double-strand (ds) DNA immobilized in a glass capillary wall and p50 proteins. For this purpose, we developed a regenerable FRET-based system comprising of a single-strand (ss) DNA with auto-complementary sequence that is end-labeled with Cy5 dye and is highly specific for p50 proteins. The proteins were labeled with a Black Hole Quencher (BHQ-3) to be used as FRET pair. The interaction of p50/p50 homodimer active form with its DNA binding site was demonstrated by both electrophoretic mobility shift assays and FRET studies. These preliminary results demonstrated the feasibility of the FRET-based DNA technique to detect the active form of NF-{kappa}B protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable.},
doi = {10.1016/j.snb.2005.07.014},
journal = {Sensors and Actuators B: Chemical},
number = 2,
volume = 113,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
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