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 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.
Selection of published references for TransAM® Nrf2
- “Physiological cyclic strain promotes endothelial cell survival via the induction of heme oxygenase-1” by Liu et al (2013) Am. J. Physiol. Heart Circ. Physiol. 304(12):H1634-H1643.
- “Angiotensin receptor-mediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats” by Vázquez-Medina et al (2013) Am. J. Physiol. Heart Circ. Physiol. 305(4):H599-H607.
- “Gestational Diabetes Mellitus Impairs Nrf2-Mediated Adaptive Antioxidant Defenses and Redox Signaling in Fetal Endothelial Cells In Utero” by Cheng et al (2013) Diabetes 62(12):4088-4097.
- “Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy” by Zhong et al (2013) Investigative Ophth. & Visual Sci. 54(6):3941-3948.
- “Prolonged fasting activates Nrf2 in post-weaned elephant seals” by Vázquez-Medina et al (2013) J. Exp. Biol. 216(15):2870-2878.
- “A defect in Nrf2 signaling constitutes a mechanism for cellular stress hypersensitivity in a genetic rat model of type 2 diabetes” by Bitar et al (2012) Am. J. Physiol. Endocrinol. Metab. 301(6):E1119-E1129.
- “Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging” by Bailey-Downs et al (2012) J Gerontol A Biol Sci Med Sci 67A(4):313-329.
- “Chronic Anthracycline Cardiotoxicity: Molecular and Functional Analysis with Focus on Nuclear Factor Erythroid 2-Related Factor 2 and Mitochondrial Biogenesis Pathways” by Jirkovsky et al (2012) Journal of Pharmacology Exp. Ther. 343(2):468-478.
- “TLR Signaling Prevents Hyperoxia-Induced Lung Injury by Protecting the Alveolar Epithelium from Oxidant-Mediated Death” by Ballinger et al (2012) The Journal of Immunology 189(1):356-364.
- “Fumarates Promote Cytoprotection of Central Nervous System Cells against Oxidative Stress via the Nuclear Factor (Erythroid-Derived 2)-Like 2 Pathway” by Scannevin et al (2012) The Journal of Pharmacology and Experimental Therapeutics 341(1):274-284.
- “Vascular oxidative stress in aging: a homeostatic failure due to dysregulation of NRF2-mediated antioxidant response” by Ungvari et al (2011) Am. J. Physiol. Heart Circ. Physiol. 301(2):H363-H372.
- “Age-Associated Vascular Oxidative Stress, Nrf2 Dysfunction, and NF-κB Activation in the Nonhuman Primate Macaca mulatta” by Ungvari et al (2011) J Gerontol A Biol Sci Med Sci 66a(8):866-875.
- “The Heme Oxygenase-1 Protein Is Overexpressed in Human Renal Cancer Cells following Activation of the Ras-Raf-ERK Pathway and Mediates Anti-Apoptotic Signal” by Banerjee et al (2011) Journal of Biological Chemistry 286(38):33580-33590.
- “Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids” by Afonyushkin et al (2011) Journal of Lipid Research 52(1):98-103.
- “Overexpression of Nrf2 Protects Cerebral Cortical Neurons from Ethanol-Induced Apoptotic Death” by Narasimhan et al (2011) Molecular Pharmacology 80(6):988-999.
- “Oxidized Phospholipids Regulate Expression of ATF4 and VEGF in Endothelial Cells via NRF2-Dependent Mechanism: Novel Point of Convergence Between Electrophilic and Unfolded Protein Stress Pathways” by Afonyushkin et al (2010) Arterioscler. Thromb. Vasc. Biol. 30(5):1007-1013.
- “Disruption of the mGsta4 Gene Increases Life Span of C57BL Mice” by Singh et al (2010) J Gerontol A Biol Sci Med Sci 65A(1):14-23.
- “CR6-interacting Factor 1 (CRIF1) Regulates NF-E2-related Factor 2 (NRF2) Protein Stability by Proteasome-mediated Degradation” by Kang et al (2010) Journal of Biological Chemistry 285(28):21258-21268.
- “NF-E2-related factor 2 regulates the stress response to UVA-1-oxidized phospholipids in skin cells” by Gruber et al (2010) The FASEB Journal 24(1):39-48.
- “Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow” by Ali et al (2009) Journal of Biological Chemistry 284(28):18882-18892.
- “Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer's disease” by Kanninen et al (2009) Proc. Natl. Acad. Sci. USA 106(38):16505-16510.
- “Introducing the ‘TCDD-Inducible AhR-Nrf2 Gene Battery’” by Yeager et al (2009) Toxicological Sciences 111(2):238-246.
- “Protective Effects of 6-(Methylsulfinyl)hexyl Isothiocyanate on Aβ1-42-Induced Cognitive Deficit, Oxidative Stress, Inflammation, and Apoptosis in Mice” by Morroni et al (2018) Int J Mol Sci 19(7): E2083.
- “The NRF2 transcriptional target, OSGIN1, contributes to monomethyl fumarate-mediated cytoprotection in human astrocytes.” by Brennan et al. (2017) Scientific Reports 7:42054.
- “Evaluation of the adaptogenic potential exerted by ginsenosides Rb1 and Rg1 against oxidative stress-mediated neurotoxicity in an in vitro neuronal model.” by Fernández-Moriano et al. (2017) PLoS One 12(8):e0182933.
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.