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Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor kappaB.

The DNA binding protein nuclear factor kappaB (NF-kappaB) 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-kappaB also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappaB 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-kappaB. 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-kappaB consensus sequence. The activated NF-kappaB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials--(i) purified recombinant NF- kappaB p50, (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-kappaB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappaB activation.