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The transcription factor NFκB (nuclear factor κB) is implicated in the regulation of many genes that code for mediators of the immune, acute phase and inflammatory responses. NFκB exists in an inactive form bound to the inhibitory IκB proteins in the cytoplasm. Treatment of cells with various inducers, such as lipopolysaccharide, TNF or IL-1, results in the degradation of the IκB proteins (See the FunctionELISA™ IκBα Kit). This releases NFκB dimers, which subsequently translocate to the nucleus where they activate appropriate target genes. Homo- and heterodimers of members of the Rel/NFκB family specifically recognize the 5´-GGGACTTTCC-3´ nucleotide sequence. The p50/p65 heterodimers and the p50 homodimers are the most common dimers found in the NFκB signaling pathway.
TransAM™ NFκB kits are available with antibodies specific for the activated forms of the p50, p52 and p65 subunits of NFκB in both colorimetric and chemiluminescent versions. The TransAM NFκB Family Kit is also available with the ability to independently screen for all the members of the NFκB family simultaneously (Figure 1). In addition, the TransAM Flexi NFκB Kits enable the study of NFκB binding at alternative sites.
Active Motif also offers recombinant NFκB p50 and p65 proteins that can be used as a standard for our TransAM NFκB Kits.
Applications
TransAM Kits can be used for studying NFκB signaling pathways in pathologies of immune response (immunodeficiencies), inflammatory response (rheumatoid arthritis, multiple sclerosis) and acute phase response (toxic shock syndromes):
- Drug screening* and/or potency
- Inhibitor or activator protein studies
- Cytokine stimulation
- Protein structure/function studies
Variety of kits and formats
- Colorimetric (p50, p52 and p65)
- Chemiluminescent (p50, p52 and p65)
- Family Kit (p50, p52, p65 (RelA), c-Rel and RelB)
- Flexi Kit (p50, p65 and Family)
Figure 1: NFκB family profiling of DNA binding activation in various cell lines.
Nuclear extracts prepared from HeLa, HeLa treated with TNF-α, Jurkat, Jurkat treated with PMA and calcium ionophore, and Raji cells were assayed at 10 µg/well for p65, p50, c-Rel, p52 and RelB activity using the TransAM NFκB Family Kit.
Figure 2: TransAM NFκB is more sensitive than gelshift.
Human fibroblast WI-38 cells are stimulated with IL-1α for 30 minutes. Increasing amounts of whole-cell extract are assayed using the TransAM NFκB p50 Kit (A) or gel retardation (B).
* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).
