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TransAM™ NFκB Chemi Kits

sensitive chemiluminescent detection of p50, p52 and p65 activation

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 form 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. In addition, the TransAM Flexi NFκB Kits enable the study of NFκB binding at alternative sites.

TransAM NFκB Chemi Kits couple the TransAM method to highly sensitive chemiluminescent detection. This format offers improved sensitivity and a greater dynamic range (Figure 1).

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 the 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 (p50, p65 and Family)

Features

Results in under 5 hours
125-fold more sensitive than Gelshift assays
Sensitivity down to 40 ng of extract/well
Quantitative results from cell or tissue extracts
High-throughput compatible
Non-radioactive assay
No cell transfections required

Figure 1: TransAM NFκB p50 Chemi sensitivity.

Nuclear extracts from Jurkat cells and Jurkat cells stimulated with TPA and calcium ionophore were assayed from 0.039 to 2.5 µg/well for NFκB p50 activation using the TransAM NFκB p50 Chemi Kit.

* 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).

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