Overview

  • Product name
    Anti-HIF-1 alpha antibody [ESEE122]
    See all HIF-1 alpha primary antibodies
  • Description
    Mouse monoclonal [ESEE122] to HIF-1 alpha
  • Host species
    Mouse
  • Specificity
    This antibody is specific for HIF-1-alpha.
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, ELISA, IHC-Fr, IHC-Pmore details
    Unsuitable for: WB
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Recombinant fragment corresponding to Human HIF-1 alpha aa 300-550.

  • Positive control
    • ICC: cultured raw mouse macrophage cells IHC-P: glioblastoma multiformae, hypoxia-induced human placenta, human normal colon
  • General notes

    This antibody clone is manufactured by Abcam.

    HIF-1 alpha can be a difficult target to work with so we have compiled a summary of all the important information you need to know including useful tips. This can be found in the protocols tab or alternatively click here to download it.

    Under normoxic conditions HIF-1 alpha has a short half-life. It is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% and upon stabilization under hypoxic conditions HIF-1 translocates to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880). Hypoxia can be induced with treatment using certain agents e.g. CoCl2 or DFO, etc. so proper sample preparation is critical.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab8366 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 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF Use a concentration of 8 - 12 µg/ml.
ELISA Use at an assay dependent concentration.
IHC-Fr 1/1000 - 1/8000.
IHC-P 1/1000 - 1/8000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
  • Application notes
    Is unsuitable for WB.
  • Target

    • Function
      Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions activates the transcription of over 40 genes, including, erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP.
    • Tissue specificity
      Expressed in most tissues with highest levels in kidney and heart. Overexpressed in the majority of common human cancers and their metastases, due to the presence of intratumoral hypoxia and as a result of mutations in genes encoding oncoproteins and tumor suppressors.
    • 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.
    • Domain
      Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
    • Post-translational
      modifications
      In normoxia, is hydroxylated on Pro-402 and Pro-564 in the oxygen-dependent degradation domain (ODD) by EGLN1/PHD1 and EGLN2/PHD2. EGLN3/PHD3 has also been shown to hydroxylate Pro-564. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Deubiquitinated by USP20. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
      In normoxia, is hydroxylated on Asn-803 by HIF1AN, thus abrogating interaction with CREBBP and EP300 and preventing transcriptional activation. This hydroxylation is inhibited by the Cu/Zn-chelator, Clioquinol.
      S-nitrosylation of Cys-800 may be responsible for increased recruitment of p300 coactivator necessary for transcriptional activity of HIF-1 complex.
      Requires phosphorylation for DNA-binding.
      Sumoylated; by SUMO1 under hypoxia. Sumoylation is enhanced through interaction with RWDD3. Desumoylation by SENP1 leads to increased HIF1A stability and transriptional activity.
      Ubiquitinated; in normoxia, following hydroxylation and interaction with VHL. Lys-532 appears to be the principal site of ubiquitination. Clioquinol, the Cu/Zn-chelator, inhibits ubiquitination through preventing hydroxylation at Asn-803.
      The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
    • Cellular localization
      Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
    • Information by UniProt
    • Database links
    • Alternative names
      • ARNT interacting protein antibody
      • ARNT-interacting protein antibody
      • Basic helix loop helix PAS protein MOP1 antibody
      • Basic-helix-loop-helix-PAS protein MOP1 antibody
      • bHLHe78 antibody
      • Class E basic helix-loop-helix protein 78 antibody
      • HIF 1A antibody
      • HIF 1alpha antibody
      • HIF-1-alpha antibody
      • HIF1 A antibody
      • HIF1 Alpha antibody
      • HIF1 antibody
      • HIF1-alpha antibody
      • HIF1A antibody
      • HIF1A_HUMAN antibody
      • Hypoxia inducible factor 1 alpha antibody
      • Hypoxia inducible factor 1 alpha isoform I.3 antibody
      • Hypoxia inducible factor 1 alpha subunit antibody
      • Hypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor antibody
      • Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor) antibody
      • Hypoxia inducible factor1alpha antibody
      • Hypoxia-inducible factor 1-alpha antibody
      • Member of PAS protein 1 antibody
      • Member of PAS superfamily 1 antibody
      • Member of the PAS Superfamily 1 antibody
      • MOP 1 antibody
      • MOP1 antibody
      • PAS domain-containing protein 8 antibody
      • PASD 8 antibody
      • PASD8 antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of hypoxia-induced Human placenta labeling HIF-1-alpha with ab8366.

    • Detection of HIF-1-alpha (red dye 568) in a cultured raw mouse macrophage cell line, using ab8366.

      Photos courtesy of Susan Alexander and Hattie Gresham, PhD.

    • Overlay histogram showing HeLa cells stained with ab8366 (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 (ab8366, 1µg/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.

    • ab8366 stained Hela cells. The cells were 4% formaldehyde fixed for 10 minutes, permeabilized in 0.1% PBS-Triton X-100 for 5 min and then blocked in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8366 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed  used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    • IHC image of HIF-1-alpha staining in human normal colon formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8366, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

       

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

      *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

    • Detection of HIF-1-alpha (red dye) in a cell cytospin from a lavage of a murine skin pouch infected with Staph Aureus, using ab8366. 100X magnification. Blue dye is DAPI nuclear staining.

      Photos courtesy of Susan Alexander and Hattie Gresham, PhD.

    • Detection of HIF-1-alpha (red dye) in a cell cytospin from a lavage of a murine skin pouch infected with Staph Aureus, using ab8366. Blue dye is DAPI nuclear staining.

      Photos courtesy of Susan Alexander and Hattie Gresham, PhD.

    • Detection of HIF-1-alpha (red dye 568) in a cultured raw mouse macrophage cell line, using ab8366. 100X magnification.

      Photos courtesy of Susan Alexander and Hattie Gresham, PhD.

    References

    This product has been referenced in:
    • La Porta S  et al. Endothelial Tie1-mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis. J Clin Invest 128:834-845 (2018). Read more (PubMed: 29355844) »
    • Ding Q  et al. Lack of endogenous parathyroid hormone delays fracture healing by inhibiting vascular endothelial growth factor-mediated angiogenesis. Int J Mol Med 42:171-181 (2018). Read more (PubMed: 29620150) »
    See all 33 Publications for this product

    Customer reviews and Q&As

    1-10 of 20 Abreviews or Q&A

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Blocking step
    Serum as blocking agent for 45 minute(s) · Concentration: 1.5% · Temperature: 25°C
    Antigen retrieval step
    Enzymatic
    Sample
    Rat Tissue sections (Gonad)
    Specification
    Gonad
    Permeabilization
    No
    Fixative
    Formaldehyde

    Abcam user community

    Verified customer

    Submitted Mar 13 2014

    Answer

    Thank you for contacting us.

    Unfortunately, because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products, and therefore I am not able to offer a test sample of any of the antibodies requested.

    However, the three of them have been tested in immunocytochemistry and immunohistochemistry with mouse samples. Therefore, they are all covered by the Abpromise guarantee.

    The Abpromise® guarantee ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase.

    To read about our Abpromise policy: https://www.abcam.com/index.html?pageconfig=abpromise

    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 inquiry.

    The placental tissue was a rather unique sample of which we know of no commercial source of and also did not get any more information form the scientist that used it.

    For an IHC positive control we recommend using glioblastoma multiformae.

    I am sorry I could not be of more help and hope this information is nevertheless helpful.

    Read More

    Answer

    Thank you for your call yesterday and for your patience while I have been in touch with the lab regarding the cytospin staining protocol for the images shown on the datasheet.

    We do not have much more information about the protocol, as the cytospin stains were done by an outside lab in 2005 and we did not receive the full protocol. The primary antibody was conjugated to a fluorophore prior to the staining, so no secondary antibody was used. We do not have information about the how the cytospin was prepared.

    I am sorry that we don't have further information regarding this protocol, but if there is anything else that we might be able to do for you please let me know and I'll be happy to help.

    Read More

    Answer

    Thank you for confirming that information.


    I have now organised for a new vial of ab8366 to be sent out to you. You can expect it to arrive tomorrow. The order number is xxxx(PO number FOCR xxxx). If you have any trouble receiving it please do let me know.


    I would be very interested to hear how you get on and if you have the time, as mentioned in my previous email, if you could share the details of the protocol you intend to usewith me by filling out the questionnaireI attached to the previous email (and re-attached to this email). Seeing some images of the staining when it worked and when it didn't would also be very helpful in investigating this case further.


    Many thanks for your cooperation and I wishyou all the best with your new experiments.

    Read More

    Answer

    Thank you very much for your phone call last week.

    HiF1 alpha is a very sensitive target it stabilizes under hypoxic conditions and gets degraded under normoxic conditions. Upon stabilization HIF-1 translocates to the nucleus so we recommend permeabilizing the sections.

    I have attached the protocol that we recommend for HIF1 alpha target staining. Although this protocol is not different than others so may be troubleshooting few steps will helps
    - Antigen retrieval always needs optimizations. 20 minutes is only a suggested antigen retrieval time for Microwave. Less than 20 minutes may leave the antigens under retrieved, leading to weak staining. More than 20 minutes may leave them over-retrieved, leading to nonspecific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 5, 10, 15, 20, 25 and 30 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
    - We would recommend using citrate buffer as antigen retrieval buffer however for optimizations purpose different buffers can be used e.g. EDTA, Tris EDTA.
    - Tissues sections: the tissue should be fixed immediately after removal; any delays actually degrades the protein.
    - We suggest incubating the section in Normal immune serum or 5% BSA for at least 1-2 hours.
    - Following publications may also help with protocol
    http://ajpheart.physiology.org/content/289/5/H2066.long
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC468666/?tool=pubmed
    Same clone number has been mentioned in following publications
    Beasley et al. 2002. Cancer Res. 62:2493-7. PMID: 11980639
    Giatromanolaki et al. 2003. J. Pathol. 200:222-8. PMID: 12754744
    Hui et al. 2002. Clin Cancer Res. 8:2595-604. PMID: 12171890
    I hope these suggestions will help. Should you have any further question please do not hesitate to contact me.

    Read More

    Answer

    Merci de nous avoir contactés. Les anticorps primaires Abcam sont livrés sans diluent ni anticorps secondaire. J'ai retrouvé la commande passée par ****. Cette commande concerne en effet 2 anticorps primaires, l'anti-HIF1 alpha antibody [ESEE122]référence ab8366 et l'anti-Carbonic Anhydrase IX antibody [SP106]référence ab105226. Le lien vers la liste des anticorps secondaires compatibles avec ab8366 (Mouse IgG1)est : https://www.abcam.com/index.html?pageconfig=searchresults&search=IgG1&pt=7&sk=targsp&sv=2&sn=Mouse&l=0&fViewMore=1 Le lien vers la liste des anticorps secondaires compatibles avec ab105226 (Rabbit IgG) est : https://www.abcam.com/index.html?pageconfig=searchresults&search=IgG&pt=7&sk=targsp&sv=2&sn=Rabbit&l=0&fViewMore=1 Vous pouvez affiner votre recherche en utilisant les filtres à gauche de la liste (application, conjugué, etc). La solution de dilution dépendra de l'application testée. J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions. Bonne et heureuse année 2012 à vouségalement.

    Read More

    Answer

    Thank you for contacting Abcam. I have had a chance to look through the data for at HIF1 alpha antibody panel (ab10625) that you had spoke of during our conversation. Of the antibodies included in this panel only ab8366 (Anti-HIF1 alpha antibody [ESEE122]) has been tested to work in IHC-Fr. Although the other antibodies may work in with this condition they have not been tested as such and therefore I cannot be confident that this product will meet your needs completely. As such I would recommend using ab8366 for your experiments. Positive controls for this product include glioblastoma multiformae and hypoxia-induced human placenta. I would also like to highly recommend ab1 (Anti-HIF1 alpha antibody [H1alpha67] - ChIP Grade). This product has been tested in IHC-IF on mouse and has been referenced in 33 publications. The Abreviews for this product may also be an excellent resource for your experiments. I've included links to both these antibodies as well as the direct link to the IHC-Fr Abreviews for Ab1 below. ab8366: https://www.abcam.com/hif1-alpha-antibody-esee122-ab8366.html ab1: https://www.abcam.com/HIF1-alpha-antibody-H1alpha67-ChIP-Grade-ab1.html ab1 reviews: https://www.abcam.com/index.html?datasheet=1&tab=abreviews&intabreviewid=9511 I hope this information helps you. I would also like you to know that these products are covered by our Abpromise®. Which ensures that you can trust our products to perform as stated on the datasheets, or we will offer a replacement, credit, or refund. The Abcam Abpromise® guarantee: • 100% Scientific and Customer Support for any product you buy from Abcam or one of our authorized distributors. • We guarantee our products work in the tested species and applications stated on the datasheet. • We will replace or refund products not performing as stated on the datasheet if reported within 6 months of purchase. • We investigate any quality concerns raised by customers to ensure our catalog contains products that perform to the highest standards. Please note these conditions to our Abpromise®: • Protocol information must be provided in order for the claim to be validated. • Antibodies tested in recombinant samples only are not guaranteed for use on endogenous samples. • “Predicted to react” information on the datasheets is provided for reference only and these species are not guaranteed. • Fast Track antibodies are covered for ELISA against the immunizing peptide only. I hope this information is helpful, but please do not hesitate to contact us with further questions.

    Read More

    Answer

    I am very sorry for the delay, and I really appreciate your patience. Unfortunately there is no publication available to prove the reactivity of ab8366 with cow. However, as the datasheet states, this product is guaranteed to work with cow species, and it is therefore covered by our guarantee Abpromise, that ensures this product should work in the species and applications indicated in the datasheet. If you had any problems when testing it in cow, we would be happy to give you scientific support and offer you a free of charge replacement, refund or credit note in the case it does not work. 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
    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 (lung)
    Specification
    lung
    Fixative
    Formaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: pH 6 citrate buffer
    Permeabilization
    No
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

    Abcam user community

    Verified customer

    Submitted Jun 04 2008

    1-10 of 20 Abreviews or Q&A

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