Anti-HIF-2-alpha antibody [ep190b] (ab8365)

Reviews (4)Q&A (18)References (48)

Overview

  • Product name
    Anti-HIF-2-alpha antibody [ep190b]
    See all HIF-2-alpha primary antibodies
  • Description
    Mouse monoclonal [ep190b] to HIF-2-alpha
  • Host species
    Mouse
  • Specificity
    This antibody is specific for HIF-2-alpha and does not cross-react with HIF-1-alpha.
  • Tested applications
    Suitable for: Flow Cyt, ELISA, IHC-Fr, IHC-P, WBmore details
  • Species reactivity
    Reacts with: Rat, Human
  • Immunogen

    Fusion protein (proprietary-tag) corresponding to Human HIF-2-alpha aa 535-631.

  • Positive control
    • Hypoxic A549 lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab8365 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
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
ELISA Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
IHC-P 1/1000 - 1/3000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/100 - 1/500. Detects a band of approximately 118 kDa (predicted molecular weight: 100 kDa).

Target

  • Function
    Transcription factor involved in the induction of oxygen regulated genes. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Regulates the vascular endothelial growth factor (VEGF) expression and seems to be implicated in the development of blood vessels and the tubular system of lung. May also play a role in the formation of the endothelium that gives rise to the blood brain barrier. Potent activator of the Tie-2 tyrosine kinase expression. Activation seems to require recruitment of transcriptional coactivators such as CREBPB and probably EP300. Interaction with redox regulatory protein APEX seems to activate CTAD.
  • Tissue specificity
    Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.
  • Involvement in disease
    Defects in EPAS1 are the cause of erythrocytosis familial type 4 (ECYT4) [MIM:611783]. ECYT4 is an autosomal dominant disorder characterized by increased serum red blood cell mass, elevated hemoglobin concentration and hematocrit, and normal platelet and leukocyte counts.
  • Sequence similarities
    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Post-translational
    modifications
    In normoxia, is probably hydroxylated on Pro-405 and Pro-531 by EGLN1/PHD1, EGLN2/PHD2 and/or EGLN3/PHD3. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
    In normoxia, is hydroxylated on Asn-847 by HIF1AN thus probably abrogating interaction with CREBBP and EP300 and preventing transcriptional activation.
    Phosphorylated on multiple sites in the CTAD.
    The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
    • Alternative names
      • Basic helix loop helix PAS protein MOP2 antibody
      • Basic-helix-loop-helix-PAS protein MOP2 antibody
      • bHLHe73 antibody
      • Class E basic helix-loop-helix protein 73 antibody
      • ECYT4 antibody
      • Endothelial PAS domain containing protein 1 antibody
      • Endothelial pas domain protein 1 antibody
      • Endothelial PAS domain-containing protein 1 antibody
      • EPAS 1 antibody
      • EPAS-1 antibody
      • EPAS1 antibody
      • EPAS1_HUMAN antibody
      • HIF 1 alpha like factor antibody
      • HIF 2 alpha antibody
      • HIF-1-alpha-like factor antibody
      • HIF-2-alpha antibody
      • HIF2-alpha antibody
      • HIF2A antibody
      • HLF antibody
      • Hypoxia inducible factor 2 alpha antibody
      • Hypoxia inducible factor 2 alpha subunit antibody
      • Hypoxia-inducible factor 2-alpha antibody
      • Member of PAS protein 2 antibody
      • Member of pas superfamily 2 antibody
      • MOP 2 antibody
      • MOP2 antibody
      • PAS domain-containing protein 2 antibody
      • PASD2 antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-2-alpha antibody [ep190b] (ab8365)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-2-alpha antibody [ep190b] (ab8365)Image from Xiang L et al., Diagn Pathol. 2012 Mar 27;7:32. Fig 5.; doi:10.1186/1746-1596-7-32; 27 March 2012, Diagnostic Pathology 2012, 7:32

      Immunohistochemical analysis of Human breast cancer tissue, staining HIF-2-alpha with ab8365.

      Antigen retrieval was performed using EDTA buffer in a pressure cooker, before being blocked with normal serum. Samples were incubated with primary antibody (1/2000) overnight at 4°C. Staining was detected using DAB.

    • Western blot - Anti-HIF-2-alpha antibody [ep190b] (ab8365)
      Western blot - Anti-HIF-2-alpha antibody [ep190b] (ab8365)

      HIF-2-alpha detected in hypoxic Human lysate using ab8365. Lane 1: normoxic A549 lysate control, lane 2: hypoxic A549 lysate.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-2-alpha antibody [ep190b] (ab8365)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-2-alpha antibody [ep190b] (ab8365)

      ab8365 at 1/300 dilution, staining HIF-2-alpha in cardiomyocytes of Human heart tissue section by Immunohistochemistry (Formalin/PFA fixed paraffin-embedded sections).

    • Flow Cytometry - Anti-HIF-2-alpha antibody [ep190b] (ab8365)
      Flow Cytometry - Anti-HIF-2-alpha antibody [ep190b] (ab8365)
      Overlay histogram showing HepG2 cells stained with ab8365 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8365, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    References

    This product has been referenced in:
    • Polívka J  et al. IDH1 mutation is associated with lower expression of VEGF but not microvessel formation in glioblastoma multiforme. Oncotarget 9:16462-16476 (2018). IHC-P ; Human . Read more (PubMed: 29662659) »
    • Zhang T  et al. Angiopoietin-like 4 promotes osteosarcoma cell proliferation and migration and stimulates osteoclastogenesis. BMC Cancer 18:536 (2018). WB ; Human . Read more (PubMed: 29739381) »
    See all 48 Publications for this product

    Publishing research using ab8365? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-10 of 22 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HIF-2-alpha antibody [ep190b]

     Excellent
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Peritonei tumor)
    Specification
    Peritonei tumor
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 19 2016

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HIF-2-alpha antibody [ep190b]

     Good
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Tumor tisse)
    Permeabilization
    No
    Specification
    Tumor tisse
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 29 2015

    Western blot abreview for Anti-HIF-2-alpha antibody [ep190b]

     Average
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    50 µg
    Gel Running Conditions
    Reduced Denaturing (4-12% acrylamide)
    Sample
    Human Cell lysate - whole cell (Renal cancer cell line)
    Specification
    Renal cancer cell line
    Blocking step
    Li-cor Blocking Buffer PBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 28 2014

    Question

    I am using Primary human cutaneous melanoma tissue. And they were paraffin
    samples. I placed sections in Sodium citrate in a plastic-screw top container
    and heat at 95C in boiling water for 30mins. Cool down in room temp for 20
    mins, and rinse sections in PBS: 3 times for 5mins.
    For the antibody dilutions: 1:25, 1:50, 1:100, 1:200. For blocking I used
    protein blocker. And the secondary antibody is provided from another company. And
    other primaries does works well with this secondary antibody.

    Read More
    Answer


    I was wondering, did you try any other antigen retrieval step, such as Tris EDTA (pH 8)? This has been successfully used by another researcher with human breast cancer tissue. I would suggest trying this.

    I am not sure how long you incubated with the primary antibody, but if overnight incubation has not yet been tried, I would suggest trying this as well.

    Read More

    Question


    I would like to know does the HIF-2a antibody comes with a guarantee
    (Anti-HIF2 alpha antibody [ep190b] (ab8365)? Can we refund the Product in case
    the antibody doesn't work? Thanks.

    Read More
    Answer

    Thank you for contacting us.

    We guarantee ab8365 to work in Flow Cyt, ELISA, IHC-Fr, IHC-P, WB with rat and human samples in accordance with our Abpromise. If you have difficulty in these applications and species, let us know and we will refund or replace the product for you within 6 months of purchase.

    https://www.abcam.com/index.html?pageconfig=resource&rid=10372&pid=10000&source=pagetrap

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Question

    No signal in IHC-Fr human samples. Used ab at 1ug/ml.
    What was the protocol used to test this ab in IHC-Fr?

    Read More
    Answer

    Thank you for your patience whilst I have been trying to contact our supplier to get the protocol they used to test ab8365 in IHC-Fr. Unfortunately they are unable to provide that information, therefore I think the best course of action is to either replace or refund the antibody for you.

    Please let me know how you would you like to continue and if you could also provide either the Abcam order number or the PO# used to purchase the antibody that would be very helpful.

    I look forward to your reply and I do apologize for the delay in responding to you.

    Read More

    Question

    Dear technical team,

    Our customer would like to purchase HIF2 alpha antibody with mouse monoclonal host to test WB and IHC-P.

    She searched ab8365 and wants to confirm it detects endogeneous HIF2 alpha.

    Check this please.

    Best regards,

    Read More
    Answer

    Thank you for your enquiry and your interest in our products.

    I can confirm that this antibody (ab8365: Anti-HIF2 alpha antibody) has been tested for IHC-Fr, IHC-P, WB, please take a look at the datasheet under tested application and the images.



    ab8365 can detect endogenous HIF2 alpha, however under hypoxia HIF2 alpha is induced and the signal for HIF2 alpha in Western blot is significantly stronger compared to normoxic conditions.

    If you need any further assistance in the future, please do not hesitate to contact me.

    Read More

    Question

      Thank you for your answer. Indeed the staining we obtained seems very specific, specially in the cytoplasm of the human kidney biopsies (formalin-fixed). Unfortunately we still don’t have photos because our microscope doesn’t have a camera and we have to take the slides to another institute.  My doubt is: if Hif-2 alpha is extremely rapidly degraded, how can it be staining in the cytoplasm? Shouldn’t I have no signal at all? Or can I assume that when the cells are in normoxia I detect HIF-2alpha in the cytoplasm?   To answer your question about the hypoxia conditions, we maintained the HEK cells in 200 µM CoCl2 for 6 hours to try to mimic hypoxia.  In attachment I’m sending the questionnaire filled out. If you have any more doubts please feel free to ask. Unfortunately this antibody has been purchased more than 6 months ago but It hadn’t been tested until two months ago because I was out from the lab. Anyway, the important is to be sure that the staining I'm having is specific for HIF-2alpha.  Best regards,     

    Read More
    Answer

    Thank you very much for your answer. I am sorry, I think I am also a little bit confused with the HIF proteins. There are indeed different studies - on particular study showing that in MEFs (i have not found yet a study on HEK cells) the localisation of HIF 2 alpha is mainly cytoplasmic and not inducible. http://mcb.asm.org/cgi/content/full/23/14/4959?view=long&pmid=12832481 The publication here below shows the HIF 2 alpha expression in liver, which seems to be also quite a lot of cytoplasmic staining. http://www.wjgnet.com/1007-9327/full/v16/i3/WJG-16-281-g001.htm Please have a look at these publications (and maybe others, as I am not a specialist with HIF 2 alpha) and let me know whether these might explain the stainings. I have also looked through the protocol provided. It seems very good to me. Can you just confirm what kind of controls you have performed to exclude unspecific staining? Have you performed an isotype control? This would indeed be the best control. I am looking forward to hear back from you with your comments.

    Read More

    Question

    To the Scientific Support team We ordered the monoclonal antibody Hif-2 alpha [ep190b], Ref: ab8365 and we tested it for immunohistochemistry on parafin human kidney bipsies and for immunofluorescence on HEK293T cells both in normoxia and hypoxia. We expected to obtain a nuclear staining but what we obtained was:  1 - in the immunohistochemistry we saw a specific staining in the cytoplasm 2- in the immunofluorescence it seems to be staining both the cytoplasm and nucleus.  I send in attachment some images of the immunofluorescence results. We would like to ask if there is something wrong with this antibody, as it should be staining the nucleus, and if it’s possible to change this antibody for another that works better.  Thank you very much,     

    Read More
    Answer

    Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results. Having reviewed this case, I would like to offer some suggestions to help optimize the results with this antibody. Indeed, HIF 2 alpha is a difficult target to work with. I suspect that the observed staining is indeed the real staining. Upon stabilization HIF translocates to the nucleus. Please also note that Hif-2 alpha is extremely rapidly degraded. Upon cellular re-oxygenation its is completely degraded in less than 1 minute. Please see also the image of the publication, where MEFs have been stained for HIF 2 alpha and substantial cytoplasmic staining is observed. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC162224/?tool=pubmed Additionally, HIF can be difficult to detect in cells or tissues grown under normoxic conditions. Its is stabilized only at O2 concentrations below 5% or with treatment using certain agents (CoCl2, DFO, etc.) so proper sample preparation is critical. Can you please specify what hypoxic conditions have been used to treat the HEK cells? I am looking forward to hear back from you with your comments on the above raised points. I have also attached a questionnaire, which I would be grateful if you could fill it out, so I could look into the problem in more detail. Can you please also attach an image of the kidney staining? Thank you very much! In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. I hope this information is helpful, and I thank you for your cooperation.  

    Read More

    Western blot abreview for Anti-HIF2 alpha antibody [ep190b]

     Good
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK 293)
    Loading amount
    20 µg
    Specification
    HEK 293
    Gel Running Conditions
    Non-reduced Denaturing (7.5)
    Blocking step
    Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 23 2011

    1-10 of 22 Abreviews or Q&A

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