Istituto di Fisiologia Clinica     
Giannetti A., Citti L., Domenici C., Tedeschi L., Baldini F., Wabuyele M. B., Vo-Dinh T. FRET-based protein-DNA binding assay for detection of active NF- B. In: Sensors and Actuators B-Chemical, vol. 113 (21) pp. 649 - 654. Special Issue - In honour of Professor Karl Cammann. Elsevier B.V, 2006.
A novel method to detect the active form of NF- 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- B protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable.
URL: http://www.sciencedirect.com/science?_ob=PublicationURL&_cdi=5283&_pubType=J&_auth=y&_acct=C000061181&_version=1&_urlVersion=0&_userid=5511047&md5=f9ea4b1856f97603b69691a18bf16c87&jchunk=120#120
Subject NF- kB
Transcription factor
Protein-DNA interaction

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