Anti-HIF-1 alpha 抗体 [H1alpha67] (ab1)

Reviews (42)Q&A (85)References (147)

製品の概要

  • 製品名

    Anti-HIF-1 alpha antibody [H1alpha67]
    HIF-1 alpha 一次抗体 製品一覧

  • 製品の詳細

    Mouse monoclonal [H1alpha67] to HIF-1 alpha

  • 由来種

    Mouse

  • アプリケーション

    適用あり: IP, ChIP, Flow Cyt, ICC/IF, WBmore details

  • 種交差性

    交差種: Mouse, Rat, Human

  • 免疫原

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

  • ポジティブ･コントロール

    • Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880) can be used as a positive control in WB.

  • 特記事項

    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 the English version and here to download the Mandarin.

    For WB, we recommend using positive control samples such as DFO or CoCl2 treated nulcear cell lysates such as ab180880. Ensure cell lysis occurs quickly (within 2 mins) if removed from hypoxia. Loading a high amount of sample (>50 µg) and addition of protease inhibitors (e.g. ab65621) may also enhance detection.

    This antibody clone is manufactured by Abcam. If you require a custom formulation or conjugation for your experiments, please contact orders@abcam.com. 

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
IP Use a concentration of 5 µg/ml.
ChIP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
ICC/IF 1/100 - 1/200.

PubMed: 25422886

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody.
WB Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).

We recommend blocking for 1 hour with 5% milk in TBST and reducing to 2% milk in TBST for the primary and secondary antibody incubation steps. For primary antibody incubation, we recommend 2 hours at room temperature.

We recommend Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody.

ターゲット情報

  • 機能

    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.

  • 組織特異性

    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.

  • 配列類似性

    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.

  • ドメイン

    Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).

  • 翻訳後修飾

    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.

  • 細胞内局在

    Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
  • Information by UniProt

  • 参照データベース

    see all

  • 別名

    • 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
    • HIF-1alpha antibody
    • HIF-alpha antibody
    • HIF1 A antibody
    • HIF1 Alpha antibody
    • HIF1 antibody
    • HIF1-alpha antibody
    • HIF1A antibody
    • HIF1A_HUMAN antibody
    • hifla 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

画像

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

    Lane 1 : HeLa nuclear extract lysate (ab150036) at 40 µg
    Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880) at 40 µg
    Lane 3 : HeLa nuclear control at 40 µg
    Lane 4 : HeLa nuclear DFO treated at 40 µg
    Lane 5 : Recombinant Human HIF-1 alpha protein (ab154478) at 0.001 µg

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 92 kDa


    Exposure time: 20 minutes


    We recommend using 5% milk in TBST as the blocking agent, decreasing to 2% milk in TBST during primary and secondary antibody incubation.

    Blots were developed with Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody

     

  • Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

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

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

    Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70°C; 10 µl 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: 110 kDa; HIF1 alpha

  • Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)This image is taken from an Abreview submitted by Mike Campa, no further information is known about this image
    Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 1/400 dilution + Human whole cell lysate (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?



    PVDF membrane was used and blocked for 16 hours in 5% milk.
  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

    ab1 staining HIF-1-alpha in MCF7 (Human breast adenocarcinoma cell line) cells treated with metformin hydrochloride ab12084, 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 24 hours in media containing different concentrations of ab120847 (metformin hydrochloride) in water, 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 2 hours 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. Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)This Image is courtesy of an anonymous Abreview

    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

  • Flow Cytometry - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Flow Cytometry - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

    Flow cytometry using ab1. HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 µg/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-Fluor® 488 dye. Unstained (black), untreated (red) and DFO treated (blue) cell traces are shown.

  • ChIP - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    ChIP - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)Image from Li X et al., Oncotarget. 2016;7(5):6000-14. Fig 5(D).; doi: 10.18632/oncotarget.6830. Reproduced under a Creative Commons Attribution 3.0 License.

    ChIP assays were performed using ab1 to verify the binding between HIF-1α and HREs. Primer 1 (P1) and primer 2 (P2) were designed to detect HREs1 and HREs2, respectively. PCR products were separated by gel electrophoresis on 2% agarose gel.

参考文献

This product has been referenced in:

  • Jin F  et al. HIF-1a-induced miR-23a~27a~24 cluster promotes colorectal cancer progression via reprogramming metabolism. Cancer Lett 440-441:211-222 (2019). Read more (PubMed: 30393198) »
  • Xu W  et al. Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF-1a signalling. J Cell Mol Med 23:1899-1907 (2019). Read more (PubMed: 30628201) »
See all 147 Publications for this product

ab1 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

レビューと Q&A

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レビューを送る 質問を送る

1-10 of 127 Abreviews or Q&A

Question

I have runned into problems with one of your antibodies for Hif 1 alpha (ab1).

It binds very unspecific and basically to the whole membrane. I dilute the antibody 1:1000 in TBST 2,5% milk.

At the moment we are blocking the PVDF-membrne with 5% milk for one hour room temperature..
Maybe it would work better by blocking with BSA?
Secondary antibodies is not a problem since they work fine with other antibodies

Read More
Answer

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 monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab1:

1) I think switching to PVDF is a good idea, sometimes when you have high background the membrane switch is a good stratgegy to reduce it.

2) We do recommend using milk with this antibody, but I see some customers that reviewed it got good results when doing an overnight blocking step rather than one hour, so I would recommend changing this step as well to reduce the background.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Question

I ordered on of your antibodies (HIF1alpha, ref. Ab1) and I would like to know what negative control (tissue) I can use to test it?

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Answer


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 levels of HIF1 alpha are minimal in normal tissues other than kidney, heart and tumours.
I would also suggest to consult the latest literature available through PubMed and other resources in order to find the most up-to-date information about this specific research area.

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Question

How much homology does the immunogen have with bovine HIF1 alpha? It works in bovine pulmonary endothelial cells treated with CoCl2.

Read More
Answer

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

ab16066 has 91% similarity.

Read More

Question

i tried in bovine and rat. In bovine worked well but in rat it did not. The ab1 did not work in either species. I ordered an antibody from santa cruz that is predicted to work in rats. Keep you informed.

Read More
Answer

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 sequence homology between human and rat for amino acid 530-826 is quite high nearly 82% which is OK for antibodies cross reactivity however we cannot be 100" sure. As per your email the antibody works for Bovine, this mean the antibody is OK perhaps it is not good for Rat. Just wondering have you done troubleshooting with rat samples, are you using hypoxia induced samples.

Thanks! I will look forward for receiving your feedback.

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Question

I have 2 abcam antibodies (ab1 and ab16066) and i am trying to blot for bovine, rat, pig, and human cell lines and tissues.
The homology link is
http://www.ncbi.nlm.nih.gov/homologene?cmd=Retrieve&db=homologene&dopt=MultipleAlignment&list_uids=1171
Can you tell me which species these antibodies are likely to work ? I prefer the use of ab16066 and I have a particular problem recognizing a rat HIF1alpha.

thank you

Read More
Answer

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 100% Abtrial discount code.

For more details about Abtrial, please click the link;

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Western blot abreview for Anti-HIF1 alpha antibody [H1alpha67] - ChIP Grade

 Excellent
Abreviews
abreview image
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
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Abcam user community

Verified customer

投稿 Jan 21 2013

Western blot abreview for Anti-HIF1 alpha antibody [H1alpha67] - ChIP Grade

 Excellent
Abreviews
abreview image
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
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Abcam user community

Verified customer

投稿 Jan 21 2013

Western blot abreview for Anti-HIF1 alpha antibody [H1alpha67] - ChIP Grade

 Excellent
Abreviews
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

投稿 Jan 18 2013

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HIF-1 alpha antibody [H1alpha67] - ChIP Grade

 Poor
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rabbit Tissue sections (cardiac tissue)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TRIS/EDTA pH 9
Permeabilization
No
Specification
cardiac tissue
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
Fixative
Formaldehyde
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Abcam user community

Verified customer

投稿 Aug 13 2018

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HIF-1 alpha antibody [H1alpha67] - ChIP Grade

 Inconclusive
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Breast cancer (necrotic area))
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/citrate
Permeabilization
Yes - Tween 20 (0.1%)
Specification
Breast cancer (necrotic area)
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: rt°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

投稿 Aug 10 2018

1-10 of 127 Abreviews or Q&A

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