Overview

  • Product name

    Anti-HIF-1 alpha antibody - C-terminal
    See all HIF-1 alpha primary antibodies
  • Description

    Rabbit polyclonal to HIF-1 alpha - C-terminal
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IP, IHC-P, ChIP, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat, Rabbit, Cow, Dog, Pig
  • Immunogen

    Recombinant fragment within Human HIF-1 alpha (C terminal). The exact sequence is proprietary.
    Database link: Q16665

  • Positive control

    • ICC/IF: CoCl2 treated HeLa and NIH/3T3 cells. WB: O2 treated HepG2 and MCF7 whole cell extracts; CoCL2 treated HepG2, NIH/3T3 and HeLa whole cell extracts.; HIF-1 alpha transfected HEK-293T whole cell extract. IHC-P: Human kidney cancer tissue. ChIP: HepG2 chromatin extract. IP: CoCl2 treated HepG2 whole cell extract.
  • General notes

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab228649 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
WB 1/500 - 1/10000. Predicted molecular weight: 93 kDa.
IP 1/100 - 1/570.
IHC-P 1/100 - 1/1000.
ChIP Use at an assay dependent concentration.
ICC/IF 1/100 - 1/1000.

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
    • 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

  • All lanes : Anti-HIF-1 alpha antibody - C-terminal (ab228649) at 1/1000 dilution

    Lane 1 : Untreated HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell extract
    Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) treated with 500 µM CoCl2 for 24 hours, whole cell extract

    Lysates/proteins at 30 µg per lane.

    Predicted band size: 93 kDa



    7.5% SDS-PAGE gel.

  • 4% paraformaldehyde-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for HIF-1 alpha (green) using ab228649 at 1/500 dilution in ICC/IF.

    Counterstain: Phalloidin, a cytoskeleton marker, at 1/100 dilution (red).

  • All lanes : Anti-HIF-1 alpha antibody - C-terminal (ab228649) at 1/500 dilution

    Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell extract
    Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) treated with 500 µM CoCl2 for 24 hours, whole cell extract

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-rabbit IgG

    Predicted band size: 93 kDa



    7.5% SDS-PAGE gel.

  • All lanes : Anti-HIF-1 alpha antibody - C-terminal (ab228649) at 1/5000 dilution

    Lane 1 : Non-transfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell extract
    Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with Flag-tagged HIF-1 alpha, whole cell extract

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-rabbit IgG

    Predicted band size: 93 kDa



    7.5% SDS-PAGE gel.

  • ChIP was performed with HepG2 (human liver hepatocellular carcinoma cell line) chromatin extract and 5 μg of either normal rabbit IgG or ab228649. The precipitated DNA was detected by PCR with primer set targeting to VEGF promoter.

  • All lanes : Anti-HIF-1 alpha antibody - C-terminal (ab228649) at 1/1000 dilution

    Lane 1 : Untreated HepG2 (human liver hepatocellular carcinoma cell line) whole cell extract
    Lane 2 : HepG2 (human liver hepatocellular carcinoma cell line) treated with 1% O2 for 24 hours, whole cell extract

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-rabbit IgG

    Predicted band size: 93 kDa



    7.5% SDS-PAGE gel.

  • HIF-1 alpha was immunoprecipitated from HepG2 (human liver hepatocellular carcinoma cell line) treated with 500 μM CoCl2 for 24 hours, whole cell extract using 5 µg of ab228649. Western blot was performed from the immunoprecipitate using ab228649.

    Lane 1: Control IgG IP in HepG2 treated with 500 μM CoCl2 for 24 hours, whole cell extracts.
    Lane 2: ab228649 IP in HepG2 treated with 500 μM CoCl2 for 24 hours, whole cell extracts.

  • 4% paraformaldehyde-fixed, treated and mock-treated NIH/3T3 (mouse embryo fibroblast cell line) cells stained for HIF-1 alpha (green) using ab228649 at 1/200 dilution in ICC/IF.

    Nuclear counterstain: Hoechst 33342 (blue).

  • All lanes : Anti-HIF-1 alpha antibody - C-terminal (ab228649) at 1/5000 dilution

    Lane 1 : Untreated HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
    Lane 2 : HepG2 (human liver hepatocellular carcinoma cell line) treated with 200 µM CoCl2 for 24 hours, whole cell lysate
    Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) treated with 500 µM CoCl2 for 24 hours, whole cell lysate

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-rabbit IgG

    Predicted band size: 93 kDa



    7.5% SDS-PAGE gel.

  • Paraffin-embedded human kidney cancer tissue stained for HIF-1 alpha using ab228649 at 1/500 dilution in immunohistochemical analysis.

  • All lanes : Anti-HIF-1 alpha antibody - C-terminal (ab228649) at 1/1000 dilution

    Lane 1 : Non-transfected, untreated MCF7 (human breast adenocarcinoma cell line) whole cell extract
    Lanes 2-3 & 5 : HIF-1 alpha shRNA-transfected, untreated MCF7 (human breast adenocarcinoma cell line) whole cell extract
    Lane 4 : HIF-1 alpha shRNA-transfected, untreated MCF7 (human breast adenocarcinoma cell line) whole cell extracts
    Lane 6 : Non-transfected MCF7 (human breast adenocarcinoma cell line) treated with 1% O2, whole cell extract
    Lanes 7-10 : HIF-1 alpha shRNA-transfected MCF7 (human breast adenocarcinoma cell line) treated with 1% O2, whole cell extract

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-rabbit IgG

    Predicted band size: 93 kDa



    7.5% SDS-PAGE gel.

  • 4% paraformaldehyde-fixed untreated (left panel) and 500 μM CoCl2 treated (right panel) HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for HIF-1 alpha (green) using ab228649 at 1/500 dilution in ICC/IF.

    Nuclear counterstain: Phalloidin, a cytoskeleton marker, at 1/100 dilution (red).

References

This product has been referenced in:

  • Tan L  et al. Preservation of alveolar ridge after tooth extraction with hypoxia-inducible factor-1a protein in a dog model. Exp Ther Med 17:2913-2920 (2019). Read more (PubMed: 30936961) »
See 1 Publication for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Cell lysate - whole cell (MCF7)
Gel Running Conditions
Reduced Denaturing (6% GEL)
Loading amount
100000 cells
Treatment
1. 20% O2, 2. 1% O2 for 16h
Specification
MCF7
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C

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Verified customer

Submitted Aug 26 2019

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