Description
Introduction: NFkB (Nuclear Factor NF-kappa-B) is a pleiotropic transcription factor that plays a role in many biological processes, including inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis. It is found as a homo- or heterodimeric complex containing the Rel-like domain containing proteins NFkB p65 (RELA/p65), RELB, NFkB1/p105, NFkB1/p50, REL and NFkB2/p52. The heterodimeric NFkB p65/p50 complex is the most abundant one. The dimers bind to kappa-B sites at their target genes, with the affinity of the interaction dependent on the subunit composition of the dimer. Furthermore, different dimers act as transcriptional activators or repressors, with the NFkB p65/p50 and p65-c-Rel complexes acting as activators. NFkB activity is controlled by several different mechanisms, including post-translational modifications, subcellular localisation and interactions with other coactivators or corepressors. NFkB complexes are held in the cytoplasm in an inactive state by interaction with members of the NFkB inhibitor (IkB) family. Typically, phosphorylation of IkB by IkB kinases (IKKs) in response to different activators leads to degradation of the inhibitor, allowing NFkB to translocate into the nucleus. The inhibitory effect of IkBs is primarily exerted through their interaction with NfKB p65. NFkB p65 is ubiquitinated leading to its proteosomal degradation, which is required for termination of the NFkB response. Phosphorylation of NFkB p65 on S536 stimulates acetylation of K310 by CBP, enhancing transcriptional activity. NFkB p65 is also acetylated at K122, enhancing DNA binding and impairing the interaction with NFKBIA. The protein is deacetylated by HDAC3.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to NF-kappaB p65. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for NF-kappaB p65 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain NF-kappaB p65, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of NF-kappaB p65 in the samples is then determined by comparing the O.D. of the samples to the standard curve