Anti-HIF-1-alpha antibody [H1alpha67] - ChIP Grade (ab463)

Overview

  • Product nameAnti-HIF-1-alpha antibody [H1alpha67] - ChIP GradeSee all HIF-1-alpha primary antibodies ...
  • Description
    Mouse monoclonal [H1alpha67] to HIF-1-alpha - ChIP Grade
  • Tested applicationsICC/IF, IP, WB, Gel supershift assays, IHC-P, ELISA, ChIP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Cow, Dog, Human, Pig, Bird, Ferret, Monkey
  • Immunogen

    Fusion protein corresponding to Human HIF-1-alpha aa 432-528.
    Database link: Q16665

  • Positive control
    • Glioblastoma multiforme.
  • 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 ab463 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
ICC/IF 1/100 - 1/500.
IP 1/10.
WB 1/500 - 1/1000.
Gel supershift assays Use at an assay dependent concentration.
IHC-P 1/100 - 1/300. Antigen retrieval is not essential but may optimise staining.

Please check attached publications for more details. 

ELISA Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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 [H1alpha67] - ChIP Grade images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung tissue sections labeling HIF-1-alpha with ab463.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Dog breast cancer tissue sections labeling HIF-1-alpha with ab463 at 1/500. Tissue was fixed with formaldehyde and blocked with a peroxidase block for 5 minutes at 25°C; antigen retrieval was by heat mediation with EDTA buffer. Samples were incubated with primary antibody (1/500 in 1% BSA + PBS) for 30 minutes at 25°C. A biotin-conjugated Mouse anti-mouse IgG polyclonal was used as the secondary antibody.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling HIF-1-alpha with ab463.

  • All lanes : Anti-HIF-1-alpha antibody [H1alpha67] - ChIP Grade (ab463)

    Lane 1 : Cobalt chloride treated COS-7 nuclear extracts
    Lane 2 : Unteated COS-7 nuclear extracts

References for Anti-HIF-1-alpha antibody [H1alpha67] - ChIP Grade (ab463)

This product has been referenced in:
  • Yu TM  et al. Expression of hypoxia-inducible factor-1a (HIF-1a) in infiltrating inflammatory cells is associated with chronic allograft dysfunction and predicts long-term graft survival. Nephrol Dial Transplant 28:659-70 (2013). IHC-P ; Human . Read more (PubMed: 23028107) »
  • Cannino G  et al. Glucose modulates respiratory complex I activity in response to acute mitochondrial dysfunction. J Biol Chem 287:38729-40 (2012). WB ; Human . Read more (PubMed: 23007390) »

See all 25 Publications for this product

Product Wall

Thank you for your email.

There are indeed several positive controls which can be used. It is noteworthy, that this protein is induced when cultured in hypoxia (with less oxygen). Please see additionnal protein information also on SwissProt h...

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Thank you for your enquiry. The antigen retrieval protocol that we cite on our datasheet is one that was highlighted to Abcam as being suitable for the preparation of Hif1a for immunohistochemistry. Unfortunately I do not have details of the applic...

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I am sorry to hear that the antibody is not working as expected. Your protocol looks fine.

What samples (type, species) are you using?

I would say that the band you are detecting might not be HIF1 alpha as the protein should run ...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - nuclear (squamous cell carcinoma SJG15 (SCCHN))
Loading amount 30 µg
Specification squamous cell carcinoma SJG15 (SCCHN)
Treatment 48h by deferroxamine at 200 uM
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Jul 31 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Dog Tissue sections (Breast cancer)
Specification Breast cancer
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: EDTA
Permeabilization No
Blocking step Novocastra Peroxidase Block as blocking agent for 5 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 13 2012

Thank you for contacting us. 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 monitoring product quality....

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Thank you for your reply.

Thank you for agreeing to test these products in IHC-P in dog samples. We look forward to receiving your Abreviews.

Please don't hesitate to contact me should you have further questions.

Thank you for your enquiry.

I am sorry to confirm that as far as we are aware, ab463 (Anti-HIF1 alpha antibody [H1alpha67-sup]) and ab55878 (Anti-Beclin 1 antibody) have never been tested with samples from dog. All tested species cross-react...

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Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from the ab463 Anti-HIF1 alpha antibodyfrom this panel.

The details you have kindly provide...

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

The details provided will enable us to investigate this case and will provide us with vita...

Read More

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