Development of a sensitive multi-well colorimetric assay for active NFkappaB.
The transcription factor nuclear factor kappaB (NFkappaB) 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 NFkappaB activation. Indeed, NFkappaB 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 NFkappaB. The presence of the DNA-bound transcription factor is then detected by anti-NFkappaB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFkappaB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.
Renard
P
P
Laboratoire de Biologie et Biochimie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium. patsy.renard@fundp.ac.be
Ernest
I
I
Houbion
A
A
Art
M
M
Le Calvez
H
H
Raes
M
M
Remacle
J
J
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D001667:Binding, Competitive; D002457:Cell Extracts; D002460:Cell Line; D003124:Colorimetry; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D015203:Reproducibility of Results; D020218:Response Elements / Q000235:genetics*; D012680:Sensitivity and Specificity
D000906:Antibodies; D002457:Cell Extracts; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D015345:Oligonucleotide Probes; D004247:DNA