期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 113, 期 2, 页码 649-654出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2005.07.014
关键词
NF-kappa B; transcription factor; FRET; protein-DNA interaction
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. (c) 2005 Elsevier B.V. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据