Anti-HIF-1-alpha [H1alpha67] antibody - ChIP Grade (ab1)

Overview

  • Product nameAnti-HIF-1-alpha [H1alpha67] antibody - ChIP GradeSee all HIF-1-alpha primary antibodies ...
  • Description
    Mouse monoclonal [H1alpha67] to HIF-1-alpha - ChIP Grade
  • Tested applicationsIHC-P, IP, ChIP, IHC-Fr, Flow Cyt, ICC/IF, WB more details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat, Sheep, Rabbit, Cow, Pig, Ferret, Monkey
  • Immunogen

    Fusion protein corresponding to Human HIF-1-alpha aa 400-550.

  • Positive control
    • This antibody gave a positive signal in whole cell (ab116322) and nuclear extracts of DFO-treated HeLa cells (ab180880). IHC-P on glioblastoma multiforme and also WB on nuclear extracts of a hypoxically or CoCl2 induced cell line such as MCF-7,PC12, 293, or MEF.

Properties

Applications

Our Abpromise guarantee covers the use of ab1 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
IP Use a concentration of 5 µg/ml.
ChIP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
WB Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).

Abcam recommends using 5% milk as the blocking agent.

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.
  • Target information above from: UniProt accession Q16665 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    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 [H1alpha67] antibody - ChIP Grade images

  • ab1 at 1/200 dilution staining HIF-1-alpha in human 293FT cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde, permeabilized in 0.5% Trition X-100 and blocked in 5% BSA for 1 hour at 25°C. The primary antibody was used at 1/200 dilution in PBS and incubated with sample at 4°C for 12 hours. An Alexa Fluor® 488 conjugated Goat polyclonal to mouse IgG was used as secondary at 1/500 dilution.

    See Abreview

  • ab1 staining HIF-1-alpha in mouse liver tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with formaldehyde and permeablized with 0.2% Triton-X100 before blocking with 2% BSA for 30 minutes at 20°C. The sample was incubated with primary antibody (1/200) for 9 hours at 4°C. An Alexa Fluor®555-conjugated Goat polyclonal to mouse IgG was used as secondary antibody at 1/200 dilution. DAPI was used to stain the cell nuclei (blue).

    See Abreview

  • All lanes : Anti-HIF-1-alpha [H1alpha67] antibody - ChIP Grade (ab1) at 5 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab150036)
    Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880)

    Lysates/proteins at 40 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 92 kDa
    Observed band size : 110 kDa (why is the actual band size different from the predicted?)


    Exposure time : 20 minutes

    Abcam recommends using 5% milk as the blocking agent, decreasing to 2% milk during primary and secondary incubation. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

  • All lanes : Anti-HIF-1-alpha [H1alpha67] antibody - ChIP Grade (ab1) at 5 µg/ml

    Lane 1 : Hela-Vehicle treated (Negative Control) Whole Cell Lysate (ab116321)
    Lane 2 : Hela-DFO treated (0.5mM, 24h) Whole Cell Lysate (ab116322)

    Lysates/proteins at 25 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 92 kDa
    Observed band size : 120 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 60 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 20 minutes
  • HIF-1-alpha was immunoprecipitated using 0.5mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5µg of Mouse monoclonal to HIF-1-alpha and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10min, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1.

    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1:20,000 dilution.

    Band: 110kDa; HIF1 alpha

  • Anti-HIF-1-alpha [H1alpha67] antibody - ChIP Grade (ab1) at 1/400 dilution + Human Cell lysate - whole cell (human lung adenocarcinoma cell line ADLC-5M2) treated for 16 hours with 100 micromolar deferoxamine (DFO) at 20 µg

    Performed under reducing conditions.

    Predicted band size : 92 kDa
    Observed band size : 120 kDa (why is the actual band size different from the predicted?)

    This image is taken from an Abreview submitted by Mike Campa, no further information is known about this image

    PVDF membrane was used and blocked for 16 hours in 5% milk.
  • ab1 staining HIF-1-alpha in MCF7 cells treated with metformin hydrochloride (ab120847), by ICC/IF. Decrease in HIF-1-alpha expression correlates with increased concentration of metformin hydrochloride, as described in literature.
    The cells were incubated at 37°C for 24h in media containing different concentrations of ab120847 (metformin hydrochloride) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab1 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Flow cytometry using ab1. HeLa cells were cultured untreated or with 1mM Deferoxamine (ab120727) for 24 hours to induce HIF-1-alpha protein levels. Cells were then trypsinized, fixed with paraformaldehyde and stained with ab1 (0.5 micrograms/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-488 dye. Unstained (black), untreated (blue) and DFO treated (red) cell traces are shown.

References for Anti-HIF-1-alpha [H1alpha67] antibody - ChIP Grade (ab1)

This product has been referenced in:
  • Harrison H  et al. Contrasting hypoxic effects on breast cancer stem cell hierarchy is dependent on ER-a status. Cancer Res 73:1420-33 (2013). IHC ; Human . Read more (PubMed: 23248117) »
  • Tripathy D  et al. Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia. Front Aging Neurosci 5:19 (2013). IHC-P ; Mouse . Read more (PubMed: 23675346) »

See all 40 Publications for this product

Product Wall

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for m...

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As indicated on the product datasheet (www.abcam.com/ab1), HIF1 alpha is expressed in most tissues with highest levels in kidney, heart and tumours. However, HIF1 alpha is also known to be an unstable molecule, therefore I would guess that the lev...

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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 Western blot
Sample Mouse Cell lysate - nuclear (NMuMG cells)
Loading amount 25 µg
Specification NMuMG cells
Treatment Check comments
Gel Running Conditions Reduced Denaturing (12)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Jan 21 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - nuclear (MCF7)
Loading amount 25 µg
Specification MCF7
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Jan 21 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Cat Cell lysate - other (cells)
Loading amount 50 µg
Specification cells
Gel Running Conditions Reduced Denaturing (10%)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Abcam user community

Verified customer

Submitted Jan 18 2013

Thank you for your kind response.

I can certainly arrange the credit note as your customer would prefer. Could you please confirm the Abcam Order Number and the date of purchase which is necessary to be able to raise a credit? This informatio...

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Application Western blot
Loading amount 70 µg
Gel Running Conditions Reduced Denaturing
Sample Rat Tissue lysate - other (Soleus (skeletal Muscle))
Specification Soleus (skeletal Muscle)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 3µg/mL · Temperature: 25°C
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Verified customer

Submitted Mar 25 2014

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