Fluorescence resonance energy transfer-based method for detection of DNA binding activities on nuclear factor κB. (July 2007)

• Record Type: Journal Article
• Authors: He, Hua-Jun; Pires, Rick; Zhu, Tie-Nian; Zhou, Anhong; Gaigalas, Adolfas K.; Zou, Sige; Wang, Lili
• Is Part Of: Biotechniques. Volume 43:Number 1(2007)
• Page Start: 93
• Page End: 98
• Publication Date: 2007-07
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000112475
• Languages: English
• ISSNs: 0736-6205

Abstract

The DNA binding protein nuclear factor κB (NF-κB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-κB also plays a key role in diseases, for example, cancer. A simple, convenient, and high-throughput detection of NF-κB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-κB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-κB consensus sequence. The activated NF-κB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials—(i) purified recombinant NF-κBp50, (ii) nuclear extracts, and (iii) whole cell lysates—we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-κB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-κB activation.