Recombinant
RabMAb

Recombinant Anti-Nrf2 (phospho S40) antibody [EP1809Y] (ab76026)

Overview

  • Product name
    Anti-Nrf2 (phospho S40) antibody [EP1809Y]
    See all Nrf2 primary antibodies
  • Description
    Rabbit monoclonal [EP1809Y] to Nrf2 (phospho S40)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Dot blot, ICC/IF, IHC-P, WB, Flow Cytmore details
    Unsuitable for: IP
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human Nrf2 (phospho S40). The exact sequence is proprietary.
    (Peptide available as ab133404)

  • Positive control
    • WB: HepG2 whole cell lysate (ab7900). IHC-P: Human tonsil, breast carcinoma, ovarian carcinoma and cervical carcinoma tissue. ICC/IF: HepG2 cells. Flow Cyt: Jurkat cells.
  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76026 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
Dot blot Use at an assay dependent concentration.
ICC/IF 1/100 - 1/250.
IHC-P 1/100 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

WB 1/5000 - 1/50000. Predicted molecular weight: 68 kDa.
Flow Cyt 1/80 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

  • Application notes
    Is unsuitable for IP.
  • Target

    • 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
      modifications
      Phosphorylation 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
    • Database links
    • Alternative names
      • erythroid derived 2 antibody
      • HEBP1 antibody
      • like 2 antibody
      • NF E2 related factor 2 antibody
      • NF-E2-related factor 2 antibody
      • NF2L2_HUMAN antibody
      • NFE2 related factor 2 antibody
      • NFE2-related factor 2 antibody
      • Nfe2l2 antibody
      • Nrf 2 antibody
      • NRF2 antibody
      • Nuclear factor (erythroid derived 2) like 2 antibody
      • Nuclear factor antibody
      • nuclear factor erythroid 2 like 2 antibody
      • Nuclear factor erythroid 2 related factor 2 antibody
      • Nuclear factor erythroid 2-related factor 2 antibody
      • Nuclear factor erythroid derived 2 like 2 antibody
      see all

    Images

    • Nrf2 was abundantly expressed in carcinomas, low grade dysplasias, and non-atypical epithelia of oral tissue.

      Representative findings of Nrf2 staining in carcinoma (left), in low grade dysplasia (middle), and in non-atypical epithelium (right).

      Corresponding PLA signals are displayed in the lower row. Scale bar; 100 µm.

      Surgical specimens were transferred to 10% buffered formalin and fixed overnight. The fixed samples were embedded in paraffin, and serially sliced into 5 µm sections. After dewaxing, sections were autoclaved at 120°C for 1 min in 10 mM sodium citrate buffer (pH 6.0), and immersed in 0.3% H2O2. They were then incubated overnight at 4°C with primary antibody to Nrf2 (diluted 1:200). The sections were rinsed with 1×PBS and incubated with the secondary antibody conjugated with horseradish peroxidase at room temperature for 1 hour. The sections were then stained with 3.3′-diaminobenzidinetetrahydrochloride (DAB) and counterstained with hematoxylin.

    • Immunofluorescence analysis of Nrf2 levels in Kaposi's sarcoma skin lesions.

      B) Healthy skin (top two rows) and KS skin tissue (bottom row) were double-stained for LANA-1 (Alexa-Fluor 594- red) and host phosphorylated pNrf2 (ab76026) (Alexa-Fluor®488 – green). DAPI was used to visualize the nuclei, and the triple merge of LANA-1, pNrf2 and DAPI is shown in the third column.

      Yellow square = enlarged area.

    • Nrf2 Translocation from cytoplasm to nucleus.

      (A) Human islets were treated with dh404 for 0.5, 1 or 2 hours. The treated and untreated samples were stained with Nrf2 antibody ab76026 (Green) and DAPI (Blue). The con-focal microscope clearly showed that the Nrf2 translocation from cytoplasm to nucleus in the dh404 treated human islet cells

    • All lanes : Anti-Nrf2 (phospho S40) antibody [EP1809Y] (ab76026) at 1/50000 dilution (purified)

      Lane 1 : untreated HepG2 cell lysate
      Lane 2 : HepG2 treated with phosphatase lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

      Predicted band size: 68 kDa
      Observed band size: 90 kDa
      why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST
      Dilution buffer: 5% NFDM/TBST

    • Immunohistochemical staining of paraffin embedded human breast carcinoma with purified ab76026 at a working dilution of 1/500. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
    • Immunofluorescence staining of HepG2 cells with purified ab76026 at a working dilution of 1/100, counter-stained with DAPI. The treated cells were treated with alkaline phosphatase for 1 h at 37°C. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab76026 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

    • Overlay histogram showing Jurkat cells fixed in 4% PFA and stained with purified ab76026 at a dilution of 1 in 80 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).
    • Dot blot analysis of Nrf2 peptides using unpurified ab76026 at 1/1000 dilution followed by Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated secondary antibody at 1/1000 dilution. Blocking and diluting buffer was 5% NFDM/TBST.

       

      Lane 1: Nrf2 (pS40) phospho peptide

      Lane 2: Nrf2 non-phospho peptide

    • Immunohistochemical analysis of paraffin-embedded human breast carcinoma using unpurified ab76026 at 1/100 dilution.

    • All lanes : Anti-Nrf2 (phospho S40) antibody [EP1809Y] (ab76026) at 1/10000 dilution (unpurified)

      Lane 1 : Untreated HepG2 (human hepatocellular carcinoma) whole cell lysates 20µg
      Lane 2 : HepG2 (human hepatocellular carcinoma) treated with Alkaline Phosphatase (AP) whole cell lysates 20µg.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

      Predicted band size: 68 kDa
      Observed band size: 100 kDa why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST, dilution buffer: 5% NFDM /TBST, exposure time: 15 seconds

    • All lanes : Anti-Nrf2 (phospho S40) antibody [EP1809Y] (ab76026) at 1/20000 dilution (unpurified)

      Lane 1 : HepG2 cell lysate
      Lane 2 : HepG2 cell lysate treated with AP

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/1000 dilution

      Predicted band size: 68 kDa
      Observed band size: 90 kDa why is the actual band size different from the predicted?

    • Unpurified ab76026 staining Nrf2 (phospho S40) in Human normal lung tissue sections by IHC-P (Formaldehyde-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% casein for 30 minutes at 4°C. Antigen retrieval was by heat mediation. Samples were incubated with primary antibody (1/50) in 1% casein for 24 hours at 4°C. An undiluted HRP-conjugated Goat polyclonal to rabbit IgG was used as the secondary antibody.

      See Abreview

    • Unpurified ab76026 showing positive staining in Breast carcinoma tissue.

    • Unpurified ab76026 showing positive staining in Cervical carcinoma tissue.

    • Unpurified ab76026 showing positive staining in Ovarian carcinoma tissue.

    • Unpurified ab76026 showing positive staining in Normal tonsil tissue.

    References

    This product has been referenced in:
    • Liu YW  et al. Neuroprotection of quercetin on central neurons against chronic high glucose through enhancement of Nrf2/ARE/glyoxalase-1 pathway mediated by phosphorylation regulation. Biomed Pharmacother 109:2145-2154 (2019). Read more (PubMed: 30551472) »
    • Jiang D  et al. Monomethyl Fumarate Protects the Retina From Light-Induced Retinopathy. Invest Ophthalmol Vis Sci 60:1275-1285 (2019). Read more (PubMed: 30924852) »
    See all 69 Publications for this product

    Customer reviews and Q&As

    1-10 of 27 Abreviews or Q&A

    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (BV2 microglia)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    70 µg
    Treatment
    5µM LPA for different time points
    Specification
    BV2 microglia
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

    Dr. Lisha Joshi

    Verified customer

    Submitted Jun 12 2019

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (pancreas)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 10 mM TRIS-EDTA pH 9 at 98°C for 20 minutes, normal pressure
    Permeabilization
    Yes - 0.04% triton 100 x in the blocking solution
    Specification
    pancreas
    Blocking step
    BSA as blocking agent for 15 minute(s) · Concentration: 2% · Temperature: 25°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Dec 13 2016

    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (lungs)
    Gel Running Conditions
    Reduced Denaturing (30 minutes RT, 10% gel)
    Loading amount
    100 µg
    Specification
    lungs
    Blocking step
    2 % Topblock in TBT-0.1% Triton 100 x as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Dec 13 2016

    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (brain)
    Gel Running Conditions
    Reduced Denaturing (10)
    Loading amount
    50 µg
    Specification
    brain
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jul 12 2016

    Application
    Western blot
    Sample
    Human Tissue lysate - whole (Brain)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    50 µg
    Specification
    Brain
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jul 12 2016

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (breast cancer cell line xenograft)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9
    Permeabilization
    No
    Specification
    breast cancer cell line xenograft
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
    Fixative
    Formaldehyde

    Abcam user community

    Verified customer

    Submitted Dec 17 2015

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Human Placenta)
    Gel Running Conditions
    Reduced Denaturing (10%)
    Loading amount
    30 µg
    Specification
    Human Placenta
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Vicky

    Verified customer

    Submitted Nov 13 2015

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Human Tissue sections (Skin)
    Permeabilization
    No
    Specification
    Skin
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: RT°C
    Fixative
    Acetone

    Abcam user community

    Verified customer

    Submitted Nov 09 2015

    Answer

    ab76026 will detect Nrf2 phosphorylated at S40 regardless of whether or not it is in the nucleus. The understanding from the literature is that under some conditions Nrf2 is phosphorylated at S40 and this leads to translocation of Nrf2 from the cytoplasm to the nucleus where it can bind to antioxidant response elements and is therefore considered "active".


    However, as "Mechanism of Checmical Activation of Nrf2" by Yun Li et al shows there can exhibit Nrf2 transactivation activity regardless of p-Nrf2 expression levels (see Figure 1). In this figure pS40 is detected independent of whether Nrf2 is activated by the inducers and it also does not change expression levels in response to the inducers. The JBC paper by Bloom and Jaiswal also argues that pS40 is not required for Nrf2 activation.


    The most common method to demonstrate Nrf2 activation is to show increased nuclear expression. This can be done by running a western blot with nuclear lysates and then using a total Nrf2 antibody (not a phospho antibody) to show increased nuclear expression. Our anti-Nrf2 antibody, ab62352, would be the best choice if you are working with a human model.


    I hope this helps and please let us know if you have any other questions.

    Read More
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (From muscle, liver, aorta)
    Gel Running Conditions
    Reduced Denaturing (4-20% gradient gel)
    Loading amount
    35 µg
    Specification
    From muscle, liver, aorta
    Blocking step
    Licor as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

    Dr. Thunder Jalili

    Verified customer

    Submitted May 14 2015

    1-10 of 27 Abreviews or Q&A

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