Renard, P., Ernest, I., Houbion, A., Art, M., Le Calvez, H., Raes, M. and Remacle, J.
Notes: A double-stranded NF-κB consensus oligonucleotide sequence containing 122bp oligonucleotide was created by PCR with a biotinylated primer at the 5´ end and the NF-κB Consensus Oligonucleotide sequence at the 3´ end. The resultant probe was bound to streptavidin-coated plates and used to screen for activated NF-κB in IL-1β-stimulated WI-38 VA13 human fibroblast cell lysates. Actively bound NF-κB was detected colorimetrically with either a rabbit anti NF-kB p50 or p65 antibody, followed by a goat anti-rabbit HRP conjugated secondary antibody and a TMB solution. Recombinant hNF-κB (p50) was used as a standard in the assay. The authors provide details for each step in the process including the specific buffers, lysate volumes, and incubation times and temperatures. The NF-κB Consensus Oligonucleotide was also used in EMSAs to confirm the results observed in the screening assay. For this assay the authors used 25μg of total protein from each lysate preparation. (2690)