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

  • 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.

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

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

  • 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:
  • Jiang L  et al. Association of PHD3 and HIF2a gene expression with clinicopathological characteristics in human hepatocellular carcinoma. Oncol Lett 15:545-551 (2018). Read more (PubMed: 29375719) »
  • Ortega MA  et al. Implication of the PI3K/Akt/mTOR Pathway in the Process of Incompetent Valves in Patients with Chronic Venous Insufficiency and the Relationship with Aging. Oxid Med Cell Longev 2018:1495170 (2018). Read more (PubMed: 30057669) »
See all 51 Publications for this product

Customer reviews and Q&As

1-10 of 22 Abreviews or Q&A

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

Abcam user community

Verified customer

Submitted Jan 19 2016

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

Abcam user community

Verified customer

Submitted Sep 29 2015

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

Abcam user community

Verified customer

Submitted Jul 28 2014

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

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

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

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

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

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
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

Abcam user community

Verified customer

Submitted Aug 23 2011

1-10 of 22 Abreviews or Q&A

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