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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™ Kits in two different formats – Original and Flexi
Active Motif offers two different formats of the TransAM Kits. The original TransAM format is perfect for assaying transcription factor binding to the consensus-binding site. These TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric or chemiluminescent readout (Figure 1).
Active Motif also offers TransAM Flexi Kits that are perfect for researchers interested in studying alternative binding sites for their transcription factor. In TransAM Flexi, the researcher synthesizes a biotinylated oligo or PCR product, which contains the transcription factor-binding site of choice. A stimulated nuclear extract is mixed with this biotinylated oligo and then transferred to the wells of a 96-well streptavidin coated plate, where the biotinylated oligo containing the activated transcription factor is captured. Next, a primary antibody specific for an epitope on the bound and active form of the transcription factor is added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 2).
Figure 1: Flow chart of the TransAM process.
Figure 2: Flow chart of the TransAM Flexi process.
* 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).
