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

Overview

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

    Synthetic phospho-peptide (Human) corresponding to residues near serine 40 of Nrf2.

  • Positive control
    • Human breast carcinoma tissue, HepG2 cell lysate
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

     

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

    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.

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

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
ICC/IF 1/100 - 1/250.
IHC-P 1/100 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

WB 1/5000 - 1/50000. Predicted molecular weight: 68 kDa.Can be blocked with Nrf2 (phospho S40) peptide (ab133404).
Flow Cyt 1/80 - 1/100.

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

  • Application notesIs unsuitable for IP.
  • Target

    • FunctionTranscription 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 specificityWidely expressed. Highest expression in adult muscle, kidney, lung, liver and in fetal muscle.
    • Sequence similaritiesBelongs to the bZIP family. CNC subfamily.
      Contains 1 bZIP domain.
    • DomainAcidic 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 localizationCytoplasm > 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

    Anti-Nrf2 (phospho S40) antibody [EP1809Y] images

    • 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
      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

    • 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).
    • 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.

    • 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.
    • 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

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

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

    This product has been referenced in:
    • Masuda Y  et al. The effect of Nrf2 pathway activation on human pancreatic islet cells. PLoS One 10:e0131012 (2015). IF ; Human . Read more (PubMed: 26110640) »
    • Kirienko NV  et al. Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 112:1821-6 (2015). WB ; Human . Read more (PubMed: 25624506) »

    See all 24 Publications for this product

    Product Wall

    Abcam guarantees this product to work in the species/application used in this Abreview.
    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
    Username

    Abcam user community

    Verified customer

    Submitted Jul 12 2016

    Abcam guarantees this product to work in the species/application used in this Abreview.
    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
    Username

    Abcam user community

    Verified customer

    Submitted Jul 12 2016

    Abcam guarantees this product to work in the species/application used in this Abreview.
    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
    Username

    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
    Username

    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
    Username

    Abcam user community

    Verified customer

    Submitted Nov 09 2015

    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 cytopla...

    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
    Username

    Dr. Thunder Jalili

    Verified customer

    Submitted May 14 2015

    Application Western blot
    Loading amount 25 µg
    Gel Running Conditions Reduced Denaturing
    Sample Human Tissue lysate - whole (liver)
    Specification liver
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
    Username

    Abcam user community

    Verified customer

    Submitted Dec 19 2014



    I believe that the Nrf2 that does enter the nucleus is acetylated and phosphorylated, which will make the protein run more slowly through the SDS PAGE gel, making its apparent molecular weight seem larger than based on theoretical amino acid ...

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    Thank you for contacting us. The IgG concentration of lot GR37038-5 is 0.114 mg/ml. For a 1 ug/ml working concentration, a dilution of 1/100 will be acceptable, or 1/114 to be more precise. Please let me know if you have any questions or any difficulty...

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