Anti-HIF-1 alpha抗体[ESEE122] (ab8366)

概述

  • 产品名称
    Anti-HIF-1 alpha抗体[ESEE122]
    参阅全部 HIF-1 alpha 一抗
  • 描述
    小鼠单克隆抗体[ESEE122] to HIF-1 alpha
  • 宿主
    Mouse
  • 特异性
    This antibody is specific for HIF-1-alpha.
  • 经测试应用
    适用于: Flow Cyt, ICC/IF, ELISA, IHC-Fr, IHC-Pmore details
    不适用于: WB
  • 种属反应性
    与反应: Mouse, Rat, Cow, Human
  • 免疫原

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

  • 阳性对照
    • ICC: cultured raw mouse macrophage cells IHC-P: glioblastoma multiformae, hypoxia-induced human placenta, human normal colon
  • 常规说明

    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.

性能

应用

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.

应用 Ab评论 说明
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.
  • 应用说明
    Is unsuitable for WB.
  • 靶标

    • 功能
      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.
    • 组织特异性
      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.
    • 序列相似性
      Contains 1 basic helix-loop-helix (bHLH) domain.
      Contains 1 PAC (PAS-associated C-terminal) domain.
      Contains 2 PAS (PER-ARNT-SIM) domains.
    • 结构域
      Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
    • 翻译后修饰
      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.
    • 细胞定位
      Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
    • Information by UniProt
    • 数据库链接
    • 别名
      • 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

    图片

    • 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.

    文献

    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

    客户评价及客户问答

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    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

    提交于 Mar 13 2014

    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

    提交于 Jun 04 2008

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

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