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Gene Regulation

TransAM® NFκB p50, p52, p65 & Family Kits

dna-binding ELISA for NFkB p50, p52, p65, c-Rel and RelB

TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets (see the list at right). Each kit includes a 96-stripwell plate in which multiple copies of a specific double-stranded oligonucleotide have been immobilized. When nuclear or whole-cell extract is added, activated transcription factor of interest binds the oligonucleotide at its consensus binding site and is quantified using the included antibody, which is specific for the bound, active form of the transcription factor being studied. For complete details, click the TransAM® Method tab below.

TransAM® NFκB Transcription Factor ELISA Kits

TransAM NFκB Kits provide everything needed to study Nuclear Factor κB (NFkB), including a positive control extract. Kits are available with antibodies specific for the activated form of p50, p52 and p65. A family kit is also available with the ability to screen the members of the NFkB family (p50, p52, p65, c-Rel and RelB). The p50 and p52 antibodies can be used with human and mouse extracts, while the p65 and RelB antibodies can be used with human, mouse and rat extracts. The c-Rel antibody can only be used with human extracts. Recombinant p50 and Recombinant p65 proteins are available separately to generate an optional protein standard curve in the TransAM NFκB Transcription Factor ELISA kits. See the NFkB Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab.

* 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).

 
Name Format Cat No. Price  
TransAM® NFκB Family 2 x 96 rxns 43296 $1,125 Buy Now
TransAM® NFκB p50 1 x 96 rxns 41096 $665 Buy Now
5 x 96 rxns 41596 $2,795 Buy Now
TransAM® NFκB p52 1 x 96 rxns 48196 $665 Buy Now
5 x 96 rxns 48696 $2,795 Buy Now
TransAM® NFκB p65 1 x 96 rxns 40096 $665 Buy Now
5 x 96 rxns 40596 $2,795 Buy Now

NFkB Transcription Factor Info

The transcription factor NFkB (NF kappa B, NF-kB, or nuclear factor kB) is widely studied due to its implication in the regulation of genes that control inflammation, cell proliferation and cell survival. NFkB is comprised of homo- or heterodimers of different subunits. These subunits are members of the structurally related Rel family of transcription factors. Five different Rel proteins (also called Rel/NFkB proteins) have been identified: p50 (NF-kB1), p52 (NF-kB2), p65 (RelA), RelB and c-Rel.

p65 (RelA), RelB and c-Rel contain a transactivation domain (TD) in their C-termini, which is required for the transport of active NFkB complexes into the nucleus. In contrast, subunits p50 and p52 do not contain transactivation domains; they are unable to transactivate on their own and must form heterodimers with RelA, RelB or c-Rel. The p50/p65 heterodimers and the p50 homodimers are the most common dimers found in the NFκB signaling pathway.

Inactive NFkB dimers are sequestered in the cytoplasm of cells by the IκB family of inhibitory proteins. Activation of NFkB by external inducers such as lipopolysaccharide, TNF or IL-1, results in the phosphorylation and 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.

For additional information on transcription factor NFkB, please click here.

 
NFkB family profiling of DNA binding activation in various cell lines 
 
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.

 
TransAM NFkB data vs. gelshift 
 
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).

The TransAM® transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

Why use TransAM® transcription factor ELISAs?

  • Up to 100-fold more sensitive than gelshift assays
  • Eliminates the use of radioactivity and the need to run gels
  • Results in less than five hours
  • Colorimetric readout enables easy, quantitative analysis with spectrophotometry at 450 nm
  • 96-stripwell format enables both high and low throughput

How TransAM® transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. 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 readout (Figure 1).

Flow chart of the TransAM DNA binding transcription factor ELISA method for measurement of activated transcription factors
Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* 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).

Contents & Storage

One, two or five NFκB 96-well assay plate(s) with plate sealer(s), NFκB p50, p52 or p65 or p50, p52, p65, c-Rel and RelB primary antibody(ies), HRP-conjugated secondary antibody, NFκB wild-type and mutated oligonucleotides, positive control cell extract, DTT, Protease Inhibitor Cocktail, Herring Sperm DNA, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing & Stop Solutions. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.

 

* 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).