dna-binding ELISA for activated Nrf2 transcription factor
TransAM Nrf2 Kits are DNA-binding ELISAs that 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. 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. The ARE Respone Element is also available as a pre-cloned transfection ready luciferase reporter vector on the LightSwitch™ Reporter System page under Synthetic Response Element Collection.
The TransAM® Method tab below provides complete method details; 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.
Monitoring Nrf2 activation with the TransAM Nrf2 Kit.
|TransAM® Nrf2||1 x 96 rxns||50296||¥115,000||Buy|
|5 x 96 rxns||50796||¥489,000||Buy|
|TransAM® Nrf2 Manual|
|Gene Regulation Products & Services Brochure|
|Tools for Disease Research|
|MSDS: Sodium Azide|
|MSDS: Sulphuric Acid|
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).
Figure 1: Flow chart of the TransAM process.
Search our database of customer publications that have used our Nuclear Extract Kit.
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.