Anti-HIF-1 alpha antibody [ESEE122] (ab8366)

Reviews (2)Q&A (10)References (38)

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: 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 the English version and here to download the Mandarin.

    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
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

      see all

    • 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
      • HIF-1alpha antibody
      • HIF-alpha antibody
      • HIF1 A antibody
      • HIF1 Alpha antibody
      • HIF1 antibody
      • HIF1-alpha antibody
      • HIF1A antibody
      • HIF1A_HUMAN antibody
      • hifla 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) - Anti-HIF-1 alpha antibody [ESEE122] (ab8366)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [ESEE122] (ab8366)

      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

    • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [ESEE122] (ab8366)
      Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [ESEE122] (ab8366)

      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.

    References

    This product has been referenced in:

    • Hao S  et al. 2-Methoxyestradiol attenuates chronic-intermittent-hypoxia-induced pulmonary hypertension through regulating microRNA-223. J Cell Physiol 234:6324-6335 (2019). Read more (PubMed: 30246291) »
    • Ribeiro GP  et al. Comparison between two programs for image analysis, machine learning and subsequent classification. Tissue Cell 58:12-16 (2019). Read more (PubMed: 31133239) »
    See all 38 Publications for this product

    Publishing research using ab8366? 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 12 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HIF-1-alpha antibody [ESEE122]

     Good
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 13 2014

    Question

    Product code: 8366

    Inquiry: Dear ladies and gentlemen, concerning the positive control to your Antibody against HIF1a (clone ESEE122), what is meant in detail by "hypoxia induced placenta"? Placenta cultivated at lox oxygen concentrations? For how long? Term placenta or first trimester placenta? Which oxygen concentration? Which part of the placenta? Thank you very much in advance for your help!

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    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.

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    Question

    Is there more information about the IF cytospin protocol for the images shown on the datasheet?

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    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.

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    Question

    ab is staining the negative tissues also
    Samples- Prostate cancer tissue sections and Normal prostate cancer tissue sections
    IHC-P application
    Microwave antigen retrieval method for 30 minutes.
    Dilution 1/10,000
    The antibody use to work previously

    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.

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    Question

    Bonjour Mme ******* de ******* (Maroc) nous a livré deux anticorps que nous avons demandés pour un projet : HIF 1 alpha et CAIX; la boite ne contient que les anticorps concentrés sans diluent et sans anticorps secondaires.Pour le diluent nous pouvons éventuellement utiliser le TBS comme c'est conseillé sur votre site mais pour l'anticorps secondaire qu'elle serait la solution?

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    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.

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    Question

    I need an anti-HIF antibody that will work in IHC-Fr on mouse tissue. I also need positive controls for this.

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    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.

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    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HIF1 alpha antibody [ESEE122]

     Good
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Jun 04 2008

    Question

    Hola buenos dias: Resulta que estoy trabajando con un anticuerpo suyo, concretamente el HIF-1 alpha (ref: ab8366, de NOVUS), dicho anticuerpo lo estamos probando en rata, pero no obtenemos resultados óptimos. Me gustaria si me pueden recomendar alguno de sus anticuerpos de HIF-1 alpha que funcione bien en rata, sobre todo para WB. Tambien, si es posible, me manden una alícuota para probarlo. Muchas gracias

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    Answer

    Thank you for your enquiry. I apologize that I am unable to answer you in Spanish. Ab8366 has not yet been tested in Western blot, so it may be this antibody is not suitable for that application. You may wish to submit an Abreview of your results from the antibody datasheet. Plese take a look at ab1 and ab2185, both of which were tested in WB and cross-react with rat. I hope this information helps. Please do not hesitate to contact us if you need anything further.

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    Question

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    Answer

    Re. our telephone conversation yesterday. The reason that lysates are not generally available to test for Hif-1 is due to the short half life of the protein. Abs can be tested using cells at 1% oxygen (hypoxic) or using brain tumour samples where hypoxic conditions are prevalent. This Ab is not generally used by customers for Western blotting and is more popular as IHC Ab, it would probably be advisable in light of this to choose one of the other Abs. One of my colleague here has also passed to me a review paper relating to Hif regulation that may be of interest to you: "Constants.A. Sending Out a Hypoxia SOS. The Scientist. 2004 Nov 22: 20-21." If you have any further questions then please get back in touch with me.

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    Question

    I have used antibody ab8366 for western blot and I have a strong band at 60 kDa. I would like to ask if the antibody also recognizes a douple band corresponding to a dimer or is it just 120 kDa the expected band.

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    Answer

    120 kDa is the expected band but the 60 kDa band is often seen in both hypoxic and normoxic cells. The same band is often seen when using ab1 as well. We do not know what the 60 kDa band represents at this time. However, using nuclear extracts decreases this background band and increases the HIF-1 alpha signal. ***Abcam does not recommend ab8366 in Western blotting ***

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    1-10 of 12 Abreviews or Q&A

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