TransAM® NFκB Chemi Kits
chemiluminescent dna-binding ELISA for activated p50, p52 and p65
TransAM® Chemi Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. 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. TransAM NFκB Chemi Kits couple the TransAM method to highly sensitive chemiluminescent detection. This format offers improved sensitivity and a greater dynamic range. For complete details, click the TransAM® Chemi tab below.
TransAM® NFκB Chemi 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. The p50 and p52 antibodies can be used with human and mouse extracts, while the p65 antibody can be used with human, mouse and rat 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.
|TransAM® NFκB p50 Chemi||1 x 96 rxns||41097||$725||Buy Now|
|5 x 96 rxns||41597||$3,025||Buy Now|
|TransAM® NFκB p52 Chemi||1 x 96 rxns||48197||$725||Buy Now|
|5 x 96 rxns||48697||$3,025||Buy Now|
|TransAM® NFκB p65 Chemi||1 x 96 rxns||40097||$725||Buy Now|
|5 x 96 rxns||40597||$3,025||Buy Now|
|TransAM® NFκB Chemi Manual|
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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.
Figure 1: TransAM NFκB p50 Chemi sensitivity.
The TransAM® Chemi 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® Chemi 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
- Luminescent readout enables easy, quantitative analysis with a luminometer or CCD camera system
- 96-stripwell format enables both high and low throughput
How TransAM® Chemi 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 Chemiluminescent Reagent to provide an easily quantified, sensitive chemiluminescent readout (Figure 1).
Figure 1: Flow chart of the TransAM Chemi process.
Contents & Storage
One or five NFκB 96-well assay plate(s) with plate sealer(s), NFκB p50, p52 or p65 primary antibody, 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 Chemiluminescent Substrate 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.