Overview

  • Product nameAnti-HIF-1-alpha antibody [mgc3]
    See all HIF-1-alpha primary antibodies
  • Description
    Mouse monoclonal [mgc3] to HIF-1-alpha
  • SpecificityThis antibody does not cross-react with ARNT or the related HIF-2-alpha.
  • Tested applicationsFlow Cyt, Immunoelectrophoresis, EMSA, Gel supershift assays, ICC/IF, IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Pig, Non Human Primates
  • Immunogen

    Recombinant fragment corresponding to Human HIF-1-alpha aa 530-826 (C terminal).

  • Positive control
    • COS-7 cells treated with 1% oxygen for 4 hours to induce hypoxia.
  • 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 ab16066 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 2µg for 106 cells.
Immunoelectrophoresis Use at an assay dependent concentration.
EMSA Use at an assay dependent concentration. PubMed: 21193390
Gel supershift assays Use at an assay dependent concentration. Only been successfully used with mouse HIF1 alpha.
ICC/IF 1/200.
IP Use at an assay dependent concentration.
WB 1/2000. Detects a band of approximately 116 kDa (predicted molecular weight: 93 kDa). 116 kDa band represents HIF-1 alpha after hypoxic induction.
IHC-P 1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. See Abreview for further information

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
    • 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 antibody
    • HIF1A_HUMAN antibody
    • Hypoxia inducible factor 1 alpha 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 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 PAS superfamily 1 antibody
    • Member of the PAS Superfamily 1 antibody
    • MOP 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 [mgc3] images

  • Immunocytochemistry/Immunofluorescent analysis of HIF-1-alpha (green) in HeLa cells either left untreated (left panel) or treated with 100uM Deferoxamine mesylate for ~16 hours (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with ab16066 at a dilution of 1/100 for at least 1 hour at room temperature and washed with PBS. Cells were then incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1/500 for 30 minutes at room temperature. F-actin (red) was stained with DyLight 594 Phalloidin and nuclei (blue) were stained with Hoechst dye.

  • Immunocytochemistry/Immunofluorescence analysis of U251 cells labeling HIF-1-alpha with ab16066. HIF-1-alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab16066 (left) at a dilution of 1/20 over night at 4°C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling HIF-1-alpha with ab16066. HIF-1 alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab16066 (left) at a dilution of 1/20 over night at 4°C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of A2058 cells labeling HIF-1-alpha with ab16066. HIF-1-alpha (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control - right) or with ab16066 (left) at a dilution of 1/20 over night at 4°C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Human fibroblasts labeling HIF-1-alpha (green) with ab16066.

    Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 1 hour at 24°C. Samples were incubated with primary antibody (1/500 in 0.3% Triton X-100 + 1% BSA) for 1 hour 30 minutes at 24°C. An AlexaFluor®488-conjugated goat anti-mouse polyclonal IgG (1/2000) was used as the secondary antibody.

    See Abreview

  • Immunocytochemistry/Immunofluorescence labeling HIF-1-alpha with ab16066.

  • ab16066 staining HIF-1-alpha in Human small intestine (IBD) and tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde; antigen retrieval was by heat mediation with an EDTA buffer (pH 9.0). Samples were incubated with primary antibody (1/800 in diluent + background reducers) for 20 minutess at 25°C. An undiluted Goat polymer was used as the secondary antibody.

  • Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 with ab16066 (left) or without primary antibody (negative control - right) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Anti-HIF-1-alpha antibody [mgc3] (ab16066) at 1/2000 dilution + HeLa cell lysate

    Predicted band size : 93 kDa
    Observed band size : 116 kDa (why is the actual band size different from the predicted?)
  • Overlay histogram showing HeLa cells stained with ab16066 (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 (ab16066, 2µg/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 [mgc3] (ab16066)

This product has been referenced in:
  • Requejo-Aguilar R  et al. PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1. Nat Commun 5:4514 (2014). Read more (PubMed: 25058378) »
  • Peng X  et al. Autophagy promotes paclitaxel resistance of cervical cancer cells: involvement of Warburg effect activated hypoxia-induced factor 1-a-mediated signaling. Cell Death Dis 5:e1367 (2014). WB ; Human . Read more (PubMed: 25118927) »

See all 12 Publications for this product

Product Wall

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (10%)
Sample Human Cell lysate - whole cell (T lymphocyte)
Specification T lymphocyte
Treatment No chemical
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Dec 12 2014

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - BD Perm/Wash Buffer
Sample Human Cell (iPSC differentiated NSCs)
Specification iPSC differentiated NSCs
Gating Strategy Reverse gate of isotype control
Preparation Cell harvesting/tissue preparation method: 50% Accutase, 50% GCDR for 5mins, inactivate with 10% FBS, and wash with PBS
Sample buffer: 10% FBS, 1% BSA
Username

Abcam user community

Verified customer

Submitted Dec 09 2014

Abcam does not currently carry anti-HIF1 alpha antibodies which are validated in flow cytometry but do not contain any carrier proteins in the storage buffer. However, we have found that many conjugation systems including our own EasyLink conjugation k...

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Abcam has not validated the combination of species/application used in this Abreview.
Application ChIP
Sample Human Cell lysate - other (HuVEC)
Specification HuVEC
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Detection step Semiquantitative PCR
Positive control Input DNA - taken from the sample before immunoprecipitating using the antibody and agarose beads
Negative control Zero antibody control
Username

Abcam user community

Verified customer

Submitted Feb 05 2013

The immunogen sequence homology of ab1 is 94% with Bovine.

ab16066 has 91% similarity.

Thank you for your email.

Please try antibody ab1 with human lysates or cells by making sure the Hypoxia is correctly induced.

ab16066 has yet to be tested with Rat samples so I am sorry we do not have any tested data available. The ...

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Thank you for contacting us.

Both of these antibodies are predicted to react with rat and other species as mentioned on the datasheet. If you are interested in testing any one of these in rat species, please let me know I am happy in sending a...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Fibroblasts)
Specification Fibroblasts
Fixative Paraformaldehyde
Permeabilization Yes - PBS+0.3% Triton-X-100
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Dr. Ravikumar Muthuswamy

Verified customer

Submitted Nov 07 2012

Do you see the same staining in your no primary antibody control? It looks like some of the more cytoplasmic staining may be background binding. It could help to add an endogenous biotin / avidin block such as ab64212.

There appear to be regi...

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Thank you for your customer's protocol. I think it is correct, and the antibody should be able to detect the protein of interest in these samples with this protocol. However, my concern is with the quality of the samples. HIF1 alpha is a transient prot...

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