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

The DNA binding protein nuclear factor KB (NF-kappa B) 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-kappa B also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappa B activation is therefore important for systematicall, 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-kappa B More specifically a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the saine end of the probe and an endonuclease binding site within the NF-kappa B consensus sequence. The activated NF-kappa B was detected by FRET following the restriction enzyme digestion. Using three different analyte materials - (i) purified recombinant NF-kappa B 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-kappa B DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappa B activation.