| Overview |
The Non-Radioactive Chemiluminescent NFκB p50/p65 Tranion Factor Assay kit is provided in a 96-well format. During the assay, the Capture Probe, a double stranded biotinylated oligonucleotide containing the flanked DNA binding consensus sequence for NFκB (5'-GGGACTTTCC-3'), is mixed with cellular (nuclear) extract in the Tranion Factor Assay Buffer provided. When incubated together, the active form of NFκB contained in the nuclear extract binds to its consensus sequence. The extract/probe/buffer mixture is then directly transferred to the streptavidin-coated plate. The active NFκB protein is immobilized on the biotinylated double stranded oligonucleotide capture probe bound to the streptavidin plate well, and any inactive, unbound material is washed away. The bound NFκB tranion factor subunits, p50 and/or p65, are detected with specific primary antibodies, a Rabbit anti-NFκB p50 and a Rabbit anti-NFκB p65. A highly sensitive HRP-conjugated secondary antibody is then used for detection. This provides sensitive chemiluminescent detection that can be read in a microplate luminometer or by a CCD camera-coupled imaging system. Included in the kit are positive cell extract, a non-specific double stranded oligonucleotide, and a specific competitor double stranded oligonucleotide. The NFκB Tranion Factor Assay was QC tested using nuclear extracts from human (HeLa) cells. Due to the conservation in the NFκB DNA binding site and the fact that the primary antibodies contained in this kit cross-react with rat and mouse NFκB, this assay is expected to work with samples from rat and mouse as well as human. |
| Background Information |
The tranion factor NF&jkappa;B (Nuclear Factor kappa B) is involved in the and regulation of a number of important cellular and physiological processes such as growth, development, apoptosis, immune and inflammatory response, and activation of various viral promoters including human immunodeficiency virus long terminal repeats1,2. NFκB represents a group of structurally related and evolutionarily conserved proteins related to the proto-oncogene c-Rel with five members in mammals that include Rel (cRel), RelA (p65), RelB, NFκB1 (p50 and its precursor p105), and NFκB2 (p52 and its precursor p100)1, 2. NFκB/Rel proteins exist as homo- or heterodimers to form tranionally competent or repressive complexes. Although most NFκB dimers are activators of tranion, the p50/50 and p52/52 homodimers can repress the tranion of their target genes. The p50/p65 heterodimer of NFκB is the most abundant in cells3. A critical component in NFκB regulation is the IκB Kinase (IKK) complex. In a majority of unstimulated cells, the NFκB tranion factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IκB proteins4,5. Upon stimulation by multiple inducers including viruses or cytokines, such as TNFα, IL-1, or PMA, I&kappaBα is rapidly phosphorylated and degraded, resulting in the release of the NFκB complex, most commonly the p105/p65 heterodimer. The p105 subunit is cleaved into its active p50 form. This cleavage exposes the NLS sequence on the p50 subunit. The p50/p65 heteroduplex then translocates to the nucleus where it activates gene tranion. NFκB induces the tranion of its own inhibitor, IκB&alapha;, causing an autoregulatory mechanism of NFκB activity and generating the inactive form of NFκB. The newly formed nuclear NFκB-IκBα complexes are then exported out to the cytoplasm, thereby reestablishing the cytoplasmic pool of inactive NFκB complexes primed for another round of activation to take place2. The wide variety of genes regulated by NFκB includes those encoding cytokines, chemokines, adhesion molecules, acute phase proteins, and inducible effector enzymes3. |
