FRET-based protein-DNA binding assay for detection of active NF-κB
A novel method to detect the active form of NF-κB, 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-κB protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable. © 2005 Elsevier B.V. All rights reserved.
Giannetti, A; Citti, L; Domenici, C; Tedeschi, L; Baldini, F; Wabuyele, MB; Vo-Dinh, T
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