RabMAb

Anti-HIF-1-alpha antibody [EP1215Y] (ab51608)

Overview

  • Product nameAnti-HIF-1-alpha antibody [EP1215Y]
    See all HIF-1-alpha primary antibodies
  • Description
    Rabbit monoclonal [EP1215Y] to HIF-1-alpha
  • Specificityab51608 recognizes HIF-1-alpha.
  • Tested applicationsICC/IF, Flow Cyt, IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human HIF-1-alpha aa 600-700 (C terminal).

  • Positive control
    • HeLa DFO-treated nuclear lysate (ab180880), Ramos cells for WB. For IHC use Human ovarian carcinoma, Rat muscule and Mouse kidney tissue sections. For ICC/IF Hela Cells(treated and untreated)
  • General notes

    We have mixed customer feedback towards the rat specificity so we are unable to confirm and guarantee its performance with rat samples. Please contact technical team for more information. Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

    A 40 μl trial size is available to purchase for this antibody.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels.

    If you have any questions regarding this update, please contact our Scientific Support team.

     

Properties

Applications

Our Abpromise guarantee covers the use of ab51608 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 Use at an assay dependent concentration. PubMed: 20846491
Flow Cyt Use at an assay dependent concentration.

For unpurified use at 1/15.

IP Use a concentration of 5 µg/ml.

For unpurified use at 5ug/mL.

WB 1/1000. Predicted molecular weight: 93 kDa.

For unpurified use at 1/100.

IHC-P 1/100.

For unpurified use at 1/15.

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 [EP1215Y] images

  • HeLa cells were untreated or treated with 1mM Deferoxamine (DFO) for 24h and fixed with paraformaldehyde for imaging by fluorescent microscopy. Cells were blocked and stained with 1X blocking buffer (ab126587). Unpurified ab51608 was used at 1:500. DAPI was used to label the nucleus. HIF1 alpha staining is absent in untreated cells and induced by DFO treatment. HIF1 alpha localizes to the nucleus.

  • Unpurified ab51608 staining HIF-1-alpha in HepG2 cells treated with baicalein (ab120723), by ICC/IF. Increase in HIF-1-alpha expression correlates with increased concentration of baicalein as described in literature.
    The cells were incubated at 37°C for 6h in media containing different concentrations of ab120723 (baicalein) 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 ab51608 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.

  • Immunohistochemical analysis using unpurified ab51608 showing positive staining in Breast carcinoma tissue.

  • Immunohistochemical analysis using unpurified ab51608 showing positive staining in Colonic adenocarcinoma tissue.

  • Immunohistochemical analysis using unpurified ab51608 showing positive staining in Squamous cell cervical carcinoma tissue.

  • All lanes : Anti-HIF-1-alpha antibody [EP1215Y] (ab51608) at 1/2000 dilution (Unpurified)

    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-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 93 kDa
    Observed band size : 110 kDa (why is the actual band size different from the predicted?)


    Exposure time : 8 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.

  • HIF-1-alpha was immunoprecipitated using 0.5mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5µg of Rabbit polyclonal to HIF1 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 unpurified ab51608.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 110kDa; HIF1 alpha


  • developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 93 kDa
    Observed band size : ~120 kDa (why is the actual band size different from the predicted?)


    Exposure time : 6 minutes

    Image courtesy of Teresa Otto (University of Duisburg-Essen, Germany)

    Anti-HIF-1-alpha unpurified antibody (ab51608) reactivity with reduced Hep3B cell lysate after transient transfection of scrambled siRNA (lanes1-3 and 7-9) or HIF-1-alpha siRNA (lanes 4-6 and 10-12). Cells were incubated at with 21% O2 (lanes 1-6) or 1% O2 (lanes 7-12) for 4h before lysis. After SDS-PAGE, membranes were blocked in 5% milk for 1h at 25°C before incubation with unpurified ab51608 (1/1,000 dilution 5% milk) for 16h at 4ºC. The blot was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.

    See Abreview

  • All lanes : Anti-HIF-1-alpha antibody [EP1215Y] (ab51608) at 1/2000 dilution (unpurified)

    Lane 1 : HeLa Whole Cell Lysate (untreated, negative control)
    Lane 2 : HeLa DFO treated (0.5mM, 24h) Whole Cell Lysate
    Lane 3 : HeLa Nuclear Cell Lysate (untreated, negative control)
    Lane 4 : HeLa Nuclear DFO treated (0.5mM, 24h) Cell Lysate

    Lysates/proteins at 40 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size : 93 kDa
    Observed band size : 110 kDa (why is the actual band size different from the predicted?)


    Exposure time : 2 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.

  • Overlay histogram showing HeLa cells stained with unpurified ab51608 (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 (ab51608, 1:50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References for Anti-HIF-1-alpha antibody [EP1215Y] (ab51608)

This product has been referenced in:
  • Zeindl-Eberhart E  et al. Epithelial-mesenchymal transition induces endoplasmic-reticulum-stress response in human colorectal tumor cells. PLoS One 9:e87386 (2014). IHC ; Human . Read more (PubMed: 24498091) »
  • Della Penna SL  et al. Renal overexpression of atrial natriuretic peptide and hypoxia inducible factor-1a as adaptive response to a high salt diet. Biomed Res Int 2014:936978 (2014). WB ; Rat . Read more (PubMed: 24689065) »

See all 16 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (7.5%, 1.5 mm)
Sample Human Cell lysate - whole cell (Hep3B)
Specification Hep3B
Treatment 1-6: 4h 21% oxygen, 7-12: 4h 1% oxygen 1-3 and 7-9: 50nM scrambled siRNA, 4-6 and 10-12: 50nM HIF-1alpha siRNA
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Dr. Teresa Otto

Verified customer

Submitted Sep 09 2013

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (glioma)
Specification glioma
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 Mar 20 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (293T and SK-Mel28)
Specification 293T and SK-Mel28
Treatment 1%O2 for 2h
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 Sep 20 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 30000 cells
Gel Running Conditions Reduced Denaturing (11%)
Sample Human Cell lysate - whole cell (MCF-7 cells (human breast adenocarcinoma cell line)
Specification MCF-7 cells (human breast adenocarcinoma cell line
Treatment 0.5% oxygen for 24hrs
Blocking step Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Sep 05 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing (8%)
Sample Human Cell lysate - whole cell (RCC4 cell line)
Specification RCC4 cell line
Blocking step Milk as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Aug 29 2013

HIF1 alpha is a difficult target to analyze and hypoxia induction is required to observe succesfully. I would suggest treating with agents such as CoCl2 or DFO(0.5mM, 24h) to increase protein levels and also prepare nuclear extracts as HIF1 alpha trans...

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Muchas gracias por tomarte la molestia de mandarnos el cuestionario y las imágenes.

He estudiado el protocolo enviado, y hay una serie de sugerencias que me gustaría haceros para intentar mejorar los resultados del anticuerpo. <...

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Gracias por ponerte en contacto con nosotros y alertarnos del problema experimentado con nuestros productos.

Cualquier comentario o queja procedente de nuestros clientes es muy importante para mejorar y monitorizar la calidad de nuestros prod...

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I am sorry we have no other HIF1 alpha antibodies that are tested in ChIP. We do have several tested in WB and IHC-P. I have checked the alignment of the immunogen of these antibodies with the zebrafish sequence. I am sorry these are all well below 70%...

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There are two isoforms of Hif1 alpha, and their predicted molecular weights based on amino acid sequences are 83 and 93 kDa- http://www.uniprot.org/uniprot/Q16665 However, isoform 1 generally runs a little over 100 kDa, and the faint band directl...

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