Recombinant Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP1808Y] to Nrf2 - ChIP Grade
- Suitable for: Flow Cyt (Intra), ChIP, ICC/IF, WB, IP, IHC-P
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Nrf2 antibody [EP1808Y] - ChIP Grade
See all Nrf2 primary antibodies -
Description
Rabbit monoclonal [EP1808Y] to Nrf2 - ChIP Grade -
Host species
Rabbit -
Specificity
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).
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Tested applications
Suitable for: Flow Cyt (Intra), ChIP, ICC/IF, WB, IP, IHC-Pmore details -
Species reactivity
Reacts with: Human -
Immunogen
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Positive control
- WB: MG-132 treated HeLa whole cell lysate, Saos-2, THP-1, MG-132 treated HepG2 whole cell lysate, MG-132 treated HCT-116 whole cell lysate. IHC-P: Human pancreatic carcinoma and kidney cancer tissues. ICC/IF: HepG2 and MG-132 treated HeLa cells. Flow Cyt (intra): HeLa cells. IP: SAOS-2 whole cell lysate. ChIP: Chromatin prepared from HepG2 cells.
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General notes
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)
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP1808Y -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Anti-Nrf2 antibody [EP1808Y] - BSA and Azide free (ab180845)
- Alexa Fluor® 488 Anti-Nrf2 antibody [EP1808Y] (ab194984)
- Alexa Fluor® 647 Anti-Nrf2 antibody [EP1808Y] (ab194985)
- HRP Anti-Nrf2 antibody [EP1808Y] (ab194986)
- Alexa Fluor® 594 Anti-Nrf2 antibody [EP1808Y] (ab206890)
- PE Anti-Nrf2 antibody [EP1808Y] (ab223926)
- APC Anti-Nrf2 antibody [EP1808Y] (ab223927)
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Assay kits
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ChIP Related Products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab62352 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) |
1/40.
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ChIP | (1) |
Use 2 µg for 25 µg of chromatin.
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ICC/IF | (4) |
1/100 - 1/500.
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WB | (24) |
1/200 - 1/1000. Predicted molecular weight: 68 kDa.
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IP |
1/20.
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IHC-P | (2) |
1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Notes |
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Flow Cyt (Intra)
1/40. |
ChIP
Use 2 µg for 25 µg of chromatin. |
ICC/IF
1/100 - 1/500. |
WB
1/200 - 1/1000. Predicted molecular weight: 68 kDa. |
IP
1/20. |
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Target
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Function
Transcription activator that binds to antioxidant response (ARE) elements in the promoter regions of target genes. Important for the coordinated up-regulation of genes in response to oxidative stress. May 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. -
Tissue specificity
Widely expressed. Highest expression in adult muscle, kidney, lung, liver and in fetal muscle. -
Sequence similarities
Belongs to the bZIP family. CNC subfamily.
Contains 1 bZIP domain. -
Domain
Acidic activation domain in the N-terminus, and DNA binding domain in the C-terminus. -
Post-translational
modificationsPhosphorylation of Ser-40 by PKC in response to oxidative stress dissociates NFE2L2 from its cytoplasmic inhibitor KEAP1, promoting its translocation into the nucleus. -
Cellular localization
Cytoplasm > cytosol. Nucleus. Cytosolic under unstressed conditions, translocates into the nucleus upon induction by electrophilic agents. - Information by UniProt
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Database links
- Entrez Gene: 4780 Human
- Omim: 600492 Human
- SwissProt: Q16236 Human
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Alternative names
- erythroid derived 2 antibody
- HEBP1 antibody
- like 2 antibody
see all
Images
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All lanes : Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352) at 1/1000 dilution
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate treated with MG-132 2uM for 18h
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 68 kDa
Observed band size: 95-110 kDa why is the actual band size different from the predicted?
Exposure time: 10 secondsBlocking and diluting buffer and concnetration: 5% NFDM/TBST.
ab181602 was used as GAPDH loading control.
The two bands are different isoforms of Nrf2, the molecular weight observed is consistent with what has been described in the literature: PMID: 17512459, PMID: 22703241.
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All lanes : Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (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
Lane 6 : Saos-2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 68 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?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] (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 CRISPR-Cas9 edited cell line ab262507 (CRISPR-Cas9 edited cell lysate ab263934). The band observed in the CRISPR-Cas9 edited 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 CRISPR-Cas9 edited 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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All lanes : Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352) at 1/1000 dilution (purified)
Lane 1 : HeLa whole cell lysate
Lane 2 : HeLa whole cell lysate treated with MG-132 2 µM for 18 hours
Lane 3 : Saos-2 whole cell lysate
Lane 4 : THP-1 whole cell lysate
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 68 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?Blocking and dilution buffer: 5% NFDM/TBST.
Exposure:
Lanes 1 and 2: 15 seconds.
Lanes 3 and 4: 3 minutes. -
Chromatin was prepared from HepG2 cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab62352 (blue), and 20µl of A/G sepharose bead slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
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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. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. The cells were co-stained with 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.
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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 (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 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) (ab150081) at 2µg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (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). -
All lanes : Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352) at 1/200 dilution (Purified)
Lane 1 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
Lane 2 : HCT 116 (Human colorectal carcinoma epithelial cell) treated with 25uM MG-132 for 4 hours whole cell lysates with 5% NFDM/TBST
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 68 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?
Exposure time: 60 secondsThe 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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreatic carcinoma tissue labelling Nrf2 with purified ab62352 at a dilution of 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
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All lanes : Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352) at 1/200 dilution (Purified)
Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell) treated with 2uM MG-132 for 18 hours whole cell lysates with 5% NFDM/TBST
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 68 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?
Exposure time: 180 secondsThe 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.
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Different batches of ab62352 were tested on Saos-2 (Human osteosarcoma epithelial) lysate at 1.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 100 kDa.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nrf2 antibody [EP1808Y] - ChIP Grade (ab62352)This image is courtesy of an abreview submitted by Rudolf Jung.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney cancer tissue sections labeling Nrf2 with ab62352 at 1/100 dilution. The tissue was fixed with paraformaldehyde and a heat mediated antigen retrival step was performed with TRIS-EDTA Buffer pH 9.0. Staining with ab62352 at 1/100 was carried out in a dilution buffer with blocking for 30 minutes at 20°C. A undiluted goat anti-rabbit HRP conjugated secondary antibody was used.
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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) (ab150077) secondary antibody (1/1000) was used as the secondary antibody. Cells were counterstained with 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.
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ab62352 (purified) at 1/30 immunoprecipitating Nrf2 in SAOS-2 whole cell lysate.
Lane 1 (input): SAOS-2 whole cell lysate (10µg)
Lane 2 (+): ab62352 + SAOS-2 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab62352 in SAOS-2 whole cell lysate.For western blotting, ab62352 was used at a dilution of 1/500 and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Blocking and dilution buffer: 5% NFDM/TBST.
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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) (ab150077) secondary antibody (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
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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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (490)
ab62352 has been referenced in 490 publications.
- Lefort S et al. De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer. Cell Death Differ 29:54-64 (2022). PubMed: 34294889
- Xu H et al. Effect of Ginsenoside Rh1 on Proliferation, Apoptosis, and Oxidative Stress in Vascular Endothelial Cells by Regulation of the Nuclear Erythroid 2-related Factor-2/Heme Oxygenase-1 Signaling Pathway. J Cardiovasc Pharmacol 79:335-341 (2022). PubMed: 34369898
- Ji R et al. Salidroside alleviates oxidative stress and apoptosis via AMPK/Nrf2 pathway in DHT-induced human granulosa cell line KGN. Arch Biochem Biophys 715:109094 (2022). PubMed: 34813774
- Li P et al. New Target of Oxidative Stress Regulation in Cochleae: Alternative Splicing of the p62/Sqstm1 Gene. J Mol Neurosci 72:830-840 (2022). PubMed: 35048235
- Zhou X et al. MST1/2 inhibitor XMU-MP-1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system. J Cell Mol Med 26:1621-1628 (2022). PubMed: 35088536