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Gene Regulation

TransAM® Flexi NFκB p50, p65 & Family Kits

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

TransAM® Flexi Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. The TransAM Flexi format enables you to look at NFκB binding sites of your own design. This kit works by combining a stimulated nuclear extract with biotinylated oligos you generated to capture activated NFκB binding. The binding reaction is then transferred to the wells of a 96-well streptavidin coated plate 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® Flexi tab below.

TransAM® Flexi NFκB Transcription Factor ELISA Kits

TransAM Flexi NFκB Kits provide everything needed to study Nuclear Factor κB (NFkB), including a positive control extract and positive control biotinylated oligonucelotide. Kits are available with antibodies specific for the activated form of p50 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® Flexi NFκB Family 2 x 96 rxns 43298 $1,125 Buy Now
TransAM® Flexi NFκB p50 1 x 96 rxns 41098 $665 Buy Now
TransAM® Flexi NFκB p65 1 x 96 rxns 40098 $665 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.

 

 TransAM NFkB Flexi data 

 
Figure 1: Measuring NFκB p65 at different binding sites.

Five µg of nuclear extract from untreated and TNF-α-treated HeLa cells were used to assay the binding affinity of NFκB p65 for four different biotinylated 50-mer oligos. Each of the 3 test oligos (Igκ, IL-8 and uPA) contained the wild-type binding site of a promoter regulated by NFκB. These sites were compared to the consensus-binding site control provided in TransAM Flexi NFκB Kits.

The TransAM® Flexi 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® Flexi 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 450nm
  • 96-stripwell format enables both high and low throughput

TransAM Flexi Kit applications include:

  • Study of variant transcription factor binding sites
  • Analysis of native promoters
  • Confirmation of chromatin immunoprecipitation (ChIP) results
  • Determination of isoform-binding affinity for NFκB

TransAM® Flexi transcription factor ELISA Kits

TransAM Flexi Kits 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 1).

TransAM Flexi flow chart
Figure 1: 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).

Contents & Storage

TransAM Flexi Kits are designed for customers interested in studying alternative binding sites of NFκB and require you to design and synthesize your own oligos of interest, which will then be coated onto the plate. Enough control biotinylated oligo, which contains the consensus-binding site used in the original TransAM NFκB Kits, is provided to perform 40 control reactions. If you are interested in looking at only the consensus site of NFκB, please use the original TransAM NFκB Kits, as they conveniently provide the oligo precoated to the plate. Flexi Kits also include one or two 96-well streptavidin-coated assay plate(s) with plate sealer(s), NFκB p50 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, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing 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).