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Nucleic Acids Research, 2001, Vol. 29, No. 4 e21
© 2001 Oxford University Press

Development of a sensitive multi-well colorimetric assay for active NF{kappa}B

Patricia Renard*, Isabelle Ernest, Andrée Houbion, Muriel Art, Hervé Le Calvez1, Martine Raes and José Remacle

Laboratoire de Biologie et Biochimie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium and 1Active Motif, 5431-C Avenida Encinas, Carlsbad, CA 92008, USA

The transcription factor nuclear factor {kappa}B (NF{kappa}B) 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 NF{kappa}B activation. Indeed, NF{kappa}B 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 NF{kappa}B. The presence of the DNA-bound transcription factor is then detected by anti-NF{kappa}B antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NF{kappa}B, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

* To whom correspondence should be addressed. Tel: +32 81 724128; Fax: +32 81 724135; Email: patsy.renard{at}fundp.ac.be


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