Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The IκBα Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by an IκBα Detection Antibody and anti-Rabbit IgG, HRP conjugated antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of total IκBα protein.
The NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins. Activation occurs via phosphorylation of IκBα at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB. IκBα phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals including inflammatory cytokines, growth factors, and chemokines. Kinases that phosphorylate IκB at these activating sites have been identified.
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