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TransAM® Nrf2

dna-binding ELISA for activated Nrf2 transcription factor

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 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® Nrf2 Transcription Factor ELISA Kits

TransAM Nrf2 Kits provide everything needed to study NF-E2-related Factor 2 (Nrf2), including a positive control extract. The kit can be used with human, mouse and rat extracts. See the Nrf2 Info tab below for kit data and more information; the kit manual can be downloaded under the Documents tab, while a selection of papers that cite the use of TransAM Nrf2 can be seen under the Publications tab.

Name Format Cat No. Price  
TransAM® Nrf2 1 x 96 rxns 50296 $665 Buy Now
5 x 96 rxns 50796 $2,795 Buy Now

Nrf2 Transcription Factor Info

NF-E2-related factor 2 (Nrf2), also known as Nuclear Factor (erythroid-derived 2)-like 2 (NFE2L2) is a critical transcription factor in oxidative stress signaling. Nrf2 is a basic leucine zipper transcription factor that binds to the antioxidant responsive element (ARE) and may serve as a master regulator in cellular defense pathways in protecting a wide variety of tissues from various toxic exposure. Nrf2 is retained in the cytoplasm under normal conditions by interaction with the inhibitor KEAP1, but following oxidative stress Nrf is released from the inhibitor and translocates to the nucleas for activation of ARE-mediated gene expression.

Figure 1: Monitoring Nrf2 activation with the TransAM Nrf2 Kit.

0.625 to 10 µg of D,L Sulforaphane treated HepG2 nuclear extract (purple bars) and untreated HepG2 nuclear extract (copper bars) were assayed per well. Data shown are the results from wells assayed in duplicate.

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).

Selection of published references for TransAM® Nrf2

Contents & Storage

One or five 96-well plate(s) with plate sealer(s), primary antibody, HRP-conjugated secondary antibody, wild-type and mutated oligonucleotides, positive control cell extract, DTT, Herring Sperm DNA, Protease Inhibitor Cocktail, Lysis, Binding, 10X Washing and 10X Antibody Binding Buffers, and Developing and 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.