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

Overview

  • Product nameAnti-HIF-1-alpha antibody [ESEE122]See all HIF-1-alpha primary antibodies ...
  • Description
    Mouse monoclonal [ESEE122] to HIF-1-alpha
  • SpecificityThis antibody is specific for HIF-1-alpha.
  • Tested applicationsFlow Cyt, ICC/IF, ELISA, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Recombinant fragment corresponding to Human HIF-1-alpha aa 329-530.

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

    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.

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

    • FunctionFunctions 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 specificityExpressed 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 similaritiesContains 1 basic helix-loop-helix (bHLH) domain.
      Contains 1 PAC (PAS-associated C-terminal) domain.
      Contains 2 PAS (PER-ARNT-SIM) domains.
    • DomainContains 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 localizationCytoplasm. 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

    Anti-HIF-1-alpha antibody [ESEE122] images

    • 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.

    • 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.

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

    • 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.

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

    This product has been referenced in:
    • Zhou Z  et al. Human rhomboid family-1 suppresses oxygen-independent degradation of hypoxia-inducible factor-1a in breast cancer. Cancer Res 74:2719-30 (2014). IHC-P ; Human . Read more (PubMed: 24648344) »
    • Kim SH  et al. Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish. Dis Model Mech 6:925-33 (2013). ICC/IF . Read more (PubMed: 23580196) »

    See all 14 Publications for this product

    Product Wall

    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
    Username

    Abcam user community

    Verified customer

    Submitted Mar 13 2014

    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. Read More

    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 recomm...

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    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 cy...

    Read More

    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 pl...

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    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 ...

    Read More

    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 al...

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    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 [...

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    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 spec...

    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
    Username

    Abcam user community

    Verified customer

    Submitted Jun 04 2008

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"