Recombinant
RabMAb

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

Overview

  • Product name
    Anti-HIF-1 alpha antibody [EP1215Y]
    See all HIF-1 alpha primary antibodies
  • Description
    Rabbit monoclonal [EP1215Y] to HIF-1 alpha
  • Host species
    Rabbit
  • Specificity
    ab51608 recognizes HIF-1-alpha. For mouse specific Hif-1-alpha rabbit monoclonal antibody, please see ab179483 (clone ID: EPR16897). ab179483 has been confirmed for mouse samples in WB.
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    within Human HIF-1 alpha aa 600-700 (C terminal). The exact sequence is proprietary.
    Database link: Q16665
    (Peptide available as ab205542)

  • Positive control
    • WB: DFO treated HeLa nuclear lysate (ab180880) and Ramos cell lysate treated with Cocl2. IHC-P: Human ovarian carcinoma, breast carcinoma, colonic adenocarcinoma and squamous cell cervical carcinoma tissues. Human gastric cancer tissue. Human CRC tumour tissue. ICC/IF: DFO treated Hela cells, Cocl2 treated HeLa cells and baicalein treated HepG2 cells. Flow Cyt: DFO treated HeLa cells. IP: DFO treated HeLa nuclear lysate.
  • General notes

    For Mouse specific Hif-1-alpha rabbit monoclonal antibody, please see ab179483 (clone ID: EPR16897).

    ab179483 has been confirmed for Mouse sample in WB.

    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.

    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 it.

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

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 1/10000.

For unpurified use at 1/15.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP Use a concentration of 5 µg/ml.
WB 1/100 - 1/1000. Predicted molecular weight: 93 kDa.Can be blocked with HIF-1 alpha peptide (ab205542).

The antibody only works in hypoxic cell and tissue lysates.

For Mouse specific Hif-1-alpha rabbit monoclonal antibody, please see ab179483 (clone ID: EPR16897).

ab179483 has been confirmed for mouse samples in WB.

IHC-P 1/100. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.

For unpurified use at 1/15.

For IHC antigen retrieval – See protocols IHC Antigen Retrieval Protocol.

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
    • 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

Images

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

    Lane 1 : MCF-7 (normoxia)
    Lane 2 : MCF-7 treated with 0.5% oxygen for 24 hours

    Lysates/proteins at 30000 cells per lane.

    Secondary
    All lanes : Polyclonal Swine anti-rabbit IgG HRP at 1/1000 dilution

    Predicted band size: 93 kDa



    Blocking buffer: 5% milk for 16 hours at 4°C. 

    See Abreview

  • Immunohistochemical analysis of Formalin-fixed paraffin-embedded human CRC tumour tissue using ab51608 for HIF-1 alpha staining. Endogenous peroxidase of sections was inhibited by 7.5% H2O2 at room temperature

    In central tumor areas of human CRCs β-catenin was typically localized at the cell membrane (A) whereas only a weak staining was observed for cytoplasmic GRP78 (B) and HIF-1 alpha staining was found to be negative (C). At the invasion front strong nuclear β-catenin was detectable indicating EMT (D, G). In corresponding regions strong cytoplasmic GRP78 expression was found (E, H). In some of the cases an intense nuclear HIF-1 alpha staining was observed (F, with hypoxia), but not in others (I, without hypoxia) (magnification 200×; scale bar: 100 µm).

  • Immunohistochemical analysis of paraffin-embedded formalin-fixed human gastric cancer tissue stained for HIF-1 alpha using ab15608 at 1/600 dilution.  Tissue sections were counterstained with Mayer's hematoxylin. Citrate buffer (pH 6.0) antigen retrieval using standard methodology

    C. HIF-1 alpha was located mainly in the nucleus of tumor cells (positive expression ×400).
    D. HIF-1 alpha original magnification ×100.

  • ab51608 staining HIF-1-alpha in HeLa cell line treated with Cocl2 by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/500). An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG(1/200) was used as the secondary antibody. Nuclei were counterstained with DAPI(right hand image).

  • ab51608 staining HIF-1-alpha in Human ovarian carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed and paraffin-embedded, antigen retrieval was by heat mediation in Tris/EDTA buffer pH9. Samples were incubated with primary antibody (1/100). An undiluted HRP-conjugated anti-rabbit IgG was used as the secondary antibody. Tissue counterstained with Hematoxylin.

  • Anti-HIF-1 alpha antibody [EP1215Y] (ab51608) at 1/100 dilution + Ramos Cells treated with Cocl2 at 10 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L), HRP- conjugated at 1/1000 dilution

    Predicted band size: 93 kDa

  • Overlay histogram showing HeLa untreated (Blue line) and HeLa treated (Red line - Deferoxamine, 1mM, 24 hours) cells stained with ab51608. 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, 1/11709 dilution) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22ºC.

    Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

  • 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

  • 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.

  • ab51608 staining of HIF-1-alpha in untreated HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/500). An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG(1/200) was used as the secondary antibody. Nuclei were counterstained with DAPI(right hand Image).

  • 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 nuclear extract lysate (ab150036)
    Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880)

    Lysates/proteins at 40 µg per lane.

    Secondary
    All lanes : 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.

  • 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
    All lanes : 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.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References

This product has been referenced in:
  • Zhao M  et al. Reactive oxygen species induce injury of the intestinal epithelium during hyperoxia. Int J Mol Med 41:322-330 (2018). Read more (PubMed: 29138796) »
  • Liang H  et al. Hypoxia induces miR-153 through the IRE1a-XBP1 pathway to fine tune the HIF1a/VEGFA axis in breast cancer angiogenesis. Oncogene N/A:N/A (2018). Read more (PubMed: 29367761) »
See all 83 Publications for this product

Customer reviews and Q&As

1-6 of 6 Q&A

Question
Answer

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 translocates to the nucleus during hypoxia.

Read More

Answer

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.

De cualquier manera, si el anticuerpo estuviera bajo garantía (en caso de haberse comprado en los últimos 6 meses), en caso de que las sugerencias no consiguieran optimizar el protocolo, estaré encantada de ofreceros un reemplazo del mismo, o un vale sin caducidad si preferís.

Este anticuerpo requiere trabajar en condiciones reductoras para reconocer la proteína. Además de calentar las muestras os recomiendo usar un agente de reducción de la proteína, como mercaptoetanol, o SDS.
Es posible que el hecho de que no obtengáis bandas en la membrana se deba a unas condiciones de bloqueo excesivas. Si tenéis la opción de bloquear con BSA al 5% durante 1 hora, y diluir los anticuerpos en 3% de BSA en lugar de leche, quizás obtengáis mejores resultados.
La mejor manera de evaluar el rendimiento del anticuerpo, y descartar un problema en las muestras es correr en paralelo con éstas un control positivo. Para este anticuerpo recomendamos un lisado de Ramos+CoCl2, o carcinoma hepático.



Por último, te agradecería que me mandaras el número de pedido del anticuerpo, para poder comprobar que no hubo ningún problema durante su envío.

Espero que estos consejos os permitan obtener mejores resultados del anticuerpo. En caso de que no sea así, por favor, no dudéis en contactarnos de nuevo para que podamos ayudaros a resolver el problema lo más rápido y eficazmente posible.

Read More

Answer

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 productos.

La información que me facilitas es muy útil para poder entender por qué el anticuerpo no ha funcionado, pero nos ayudaría mucho que nos ampliaras la información referente al protocolo seguido.

Te envío un cuestionario que contiene todas algunas preguntas, y si pudieras, además del cuestionario completo, enviarnos una imagen nos seria de gran utilidad para intentar resolver el problema.

Nuestra garantía Abpromise garantiza que los productos funcionan tal y como se especifica en su ficha técnica, y en caso contrario, estaremos encantados de ofrecer soporte científico que ayude a mejorar los resultados. Si aun así el producto sigue sin funcionar correctamente, y éste se adquirió en los últimos seis meses, estaremos encantados de reemplazar o reembolsar el valor del anticuerpo.

Gracias por tu comprensión, y quedo a la espera de tu respuesta.

Read More

Answer

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%, and regrettably  we would not predict that they can detect in zebrafish. As previously discussed, I would be pleased to provide an Abreview testing discount if the customer would like to try one of our HIF1 alpha abs in Zebrafish samples. This will give the customer a free ab after submitting an Abreview.

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Question
Answer

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 directly underneath is most likely isoform 2. According to the lab, the weaker bands lower on the blot are seen in their testing, but they can be reduced by diluting the antibody further. I would recommend trying the antibody at 1:1000 overnight at 4C to diminish these bands. You can also include 1% BSA in the antibody diluent to reduce non-specific binding. We do guarantee all of our products to work in tested species and applications for up to 6 months after purchase, so if these suggestions do not improve the results we would be happy to send a replacement or issue a credit or refund.

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Question
Answer

The concentration of the lot you have, GR28000-3, is 0.454 mg/mL.

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