Development of a sensitive multi-well colorimetric assay for active NFκB

The transcription factor nuclear factor κB (NFκB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFκB activation. Indeed, NFκB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFκB. The presence of the DNA-bound transcription factor is then detected by anti-NFκB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFκB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.