Knockout Tested Rabbit Recombinant Monoclonal Nrf2 antibody. Suitable for WB, ICC/IF, Flow Cyt (Intra) and reacts with Human samples. Cited in 584 publications.
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
IP | ChIP | WB | ICC/IF | Flow Cyt (Intra) | IHC-P | |
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Human | Not recommended | Not recommended | Tested | Tested | Tested | Not recommended |
Species | Dilution info | Notes |
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Species Human | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
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Species Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
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Species Human | Dilution info 1/200 - 1/1000 | Notes - |
Species | Dilution info | Notes |
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Species Human | Dilution info 1/1000 - 1/5000 | Notes - |
Species | Dilution info | Notes |
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Species Human | Dilution info 1/40 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
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Transcription factor that plays a key role in the response to oxidative stress: binds to antioxidant response (ARE) elements present in the promoter region of many cytoprotective genes, such as phase 2 detoxifying enzymes, and promotes their expression, thereby neutralizing reactive electrophiles (PubMed:11035812, PubMed:19489739, PubMed:29018201, PubMed:31398338). In normal conditions, ubiquitinated and degraded in the cytoplasm by the BCR(KEAP1) complex (PubMed:11035812, PubMed:15601839, PubMed:29018201). In response to oxidative stress, electrophile metabolites inhibit activity of the BCR(KEAP1) complex, promoting nuclear accumulation of NFE2L2/NRF2, heterodimerization with one of the small Maf proteins and binding to ARE elements of cytoprotective target genes (PubMed:19489739, PubMed:29590092). The NFE2L2/NRF2 pathway is also activated in response to selective autophagy: autophagy promotes interaction between KEAP1 and SQSTM1/p62 and subsequent inactivation of the BCR(KEAP1) complex, leading to NFE2L2/NRF2 nuclear accumulation and expression of cytoprotective genes (PubMed:20452972). May also be involved in the transcriptional activation of genes of the beta-globin cluster by mediating enhancer activity of hypersensitive site 2 of the beta-globin locus control region (PubMed:7937919).
Nuclear factor erythroid 2-related factor 2, NF-E2-related factor 2, NFE2-related factor 2, Nrf-2, HEBP1, NRF2, NFE2L2
Knockout Tested Rabbit Recombinant Monoclonal Nrf2 antibody. Suitable for WB, ICC/IF, Flow Cyt (Intra) and reacts with Human samples. Cited in 584 publications.
Nuclear factor erythroid 2-related factor 2, NF-E2-related factor 2, NFE2-related factor 2, Nrf-2, HEBP1, NRF2, NFE2L2
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EP1808Y
Affinity purification Protein A
The expression of Nrf2 is stimulated by oxidative stress, electrophiles and chemical activators (PMID: 25761198, PMID: 27638861 and PMID: 28587109). Nrf2 antibody (ab62352) detects no signal in most untreated samples in WB. Stimuli treated samples are recommended. We do not recommend using this product in western blot with tissue lysates, however some customers have used this antibody successfully using concentrated samples (see submitted abreviews).
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
PLEASE NOTE:
Nrf2 antibody (ab62352) detects no signal in most untreated samples for WB. Stimuli treated samples are recommended. Nrf2 expression is stimulated by oxidative stress, electrophiles and chemical activators (PMID: 25761198, PMID: 27638861 and PMID: 28587109
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
This supplementary information is collated from multiple sources and compiled automatically.
Nrf2 also known as nuclear factor erythroid 2-related factor 2 is a transcription factor with a molecular weight of approximately 66 kDa. It plays a mechanical role in regulating the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. Nrf2 is ubiquitously expressed in various tissues including the liver lungs and skin. Nrf2 activation occurs when it translocates from the cytoplasm to the nucleus to bind the antioxidant response element (ARE) in the DNA starting transcription of target genes.
Nrf2 acts as an important regulator of the cellular antioxidant response. It works in conjunction with its partner protein Keap1 forming a complex that controls its stability and degradation. Under normal conditions Keap1 keeps Nrf2 in the cytoplasm where it is targeted for ubiquitination and degradation. Once activated by oxidative stress or electrophiles Nrf2 dissociates from Keap1 thereby avoiding degradation and relocates to the nucleus to activate the transcription of ARE-dependent genes. This activity boosts the cellular response to oxidative stress by inducing genes involved in detoxification and cellular defense.
Nrf2 plays a significant role in the oxidative stress response and detoxification pathways. Nrf2 activation is linked closely to the PI3K/Akt signaling pathway which influences cell survival growth and metabolism. This pathway also interacts with other important proteins like GSK-3β which can modulate Nrf2 activity and stability. Through these pathways Nrf2 orchestrates a defense mechanism against reactive oxygen species (ROS) by boosting the expression of antioxidant enzymes and detoxifying proteins.
Nrf2 has been associated with conditions like cancer and neurodegenerative diseases. In cancer aberrant Nrf2 activation may lead to enhanced tumor survival by increasing expression of cytoprotective genes which makes cancer cells resistant to chemotherapy. Nrf2 also interacts with proteins such as p53 which play roles in tumor suppression and cellular stress responses. In neurodegenerative disorders reduced Nrf2 activity can contribute to oxidative stress leading to neuron damage and disease progression with proteins like amyloid-beta also being linked with oxidative processes affected by Nrf2 functionality.
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This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
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ab62352 staining Nrf2 in untreated HeLa cells (top panel) and treated HeLa cells (bottom panel). Cells were treated with 2μM of MG-132 for 18 hours (MG-132, proteasome inhibitor ab141003). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab62352 at 1μg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
The expression of Nrf2 is stimulated by oxidative stress, electrophiles and chemical activators (PMID: 25761198, PMID: 27638861 and PMID: 28587109). ab62352 detects no signal in most of the untreated samples in WB. Stimuli treated samples are recommended.
All lanes: Western blot - Anti-Nrf2 antibody [EP1808Y] (ab62352) at 1/200 dilution
Lane 1: HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates at 20 µg
Lane 2: HCT 116 (Human colorectal carcinoma epithelial cell) treated with 25uM MG-132 for 4 hours whole cell lysates at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 68 kDa
Observed band size: 100 kDa
Exposure time: 60s
Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Nrf2 with purified ab62352 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. The cells were co-stained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889, an Alexa Fluor® 594-conjugated mouse anti-alpha tubulin antibody (1/200). Nuclei counterstained with DAPI (blue).
Secondary antibody only control: PBS was used instead of the primary antibody as the negative control.
Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Nrf2 with purified ab62352 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody (1/1000) was used as the secondary antibody. Cells were counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). DAPI was used to stain the nuclei blue.
Secondary antibody only control: PBS was used instead of the primary antibody as the negative control.
Intracellular Flow Cytometry analysis of HeLa cells labelling Nrf2 with purified ab62352 at a dilution of 1/60 (red). Cells were fixed with 4% paraformaldehyde. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Nrf2 with ab62352 at 1/40 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluorr®488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
Western blot analysis using ab62352 at 1:1000 on Human iPSC-cardiomyocytes. Blocking agent and dilution buffer was 5% Skim Milk in TBS-Tween.
A cellular fractionation in Human iPSC-cardiomyocyte cells was performed to separate the nucleus from the cytoplasm (Lane 2).
All lanes: Western blot - Anti-Nrf2 antibody [EP1808Y] (ab62352) at 1/1000 dilution
Lane 1: Human iPSC-cardiomyocytes cytoplasmatic fraction, at 20 µg
Lane 2: Human iPSC-cardiomyocytes nuclear fraction at 20 µg
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab6721) at 1/2000 dilution
Observed band size: 100 kDa
False colour image of Western blot: Anti-Nrf2 antibody [EP1808Y] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab62352 was shown to bind specifically to Nrf2.
Target band (indicated by arrow) was observed at 85-90 kDa in wild-type A549 cell lysates with no signal observed at this size in NFE2L2 knockout cell line Human NFE2L2 knockout A549 cell line[21] ab285359 (knockout cell lysate ab289682). A band lower than the target was present on both WT and KO, we are unsure about the identity of this band.
To generate this image, wild-type and NFE2L2 knockout A549 cell lysates were analysed. Please note that MG132 treatment does not affect expression levels of Nrf2. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352) at 1/1000 dilution
Lane 1: Wild-type A549 Vehicle control MG132 (0 uM, 18 h) cell lysate at 20 µg
Lane 2: Wild-type A549 Treated MG132 (2 uM, 18 h) cell lysate at 20 µg
Lane 3: NFE2L2 [21] knockout A549 Vehicle control MG132 (0 uM, 18 h) cell lysate at 20 µg
Lane 4: NFE2L2 [21] knockout A549 Treated MG132 (2 uM, 18 h) cell lysate at 20 µg
All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 68 kDa
False colour image of Western blot: Anti-Nrf2 antibody [EP1808Y] - ChIP Grade staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab62352 was shown to bind specifically to Nrf2. A band was observed at 85 kDa in wild-type HeLa cell lysates with no signal observed at this size in NFE2L2 knockout cell line Human NFE2L2 (Nrf2) knockout HeLa cell line ab262507 (knockout cell lysate Human NFE2L2 (Nrf2) knockout HeLa cell lysate ab263934). The band observed in the knockout lysate lane below 85 kDa is likely to represent a truncated form of Nrf2. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and NFE2L2 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-Nrf2 antibody [EP1808Y] (ab62352) at 1/500 dilution
Lane 1: Wild-type HeLa control MG132 (0 uM, 18 h) cell lysate
Lane 2: Wild-type HeLa treated MG132 (2 uM, 18 h) cell lysate
Lane 3: NFE2L2 knockout HeLa control MG132 (0 uM, 18 h) cell lysate
Lane 4: NFE2L2 knockout HeLa treated MG132 (2 uM, 18 h) cell lysate
Lane 5: HCT 116 cell lysate
ab62352 staining Nrf2 in untreated wild type A549 cells (left panel), treated wild type A549 cells (middle panel) and untreated NFE2L2 knockout A549 cells (right panel). Cells were treated with 2µM of MG-132 for 18 hours (MG-132, proteasome inhibitor ab141003). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab62352 at 0.2 µg/ml concentration and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2µg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2µg/ml (shown in magenta). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
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