Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

Reviews (10)Q&A (34)References (123)

Overview

  • Product name

    Anti-HIF-1 alpha antibody - ChIP Grade
    See all HIF-1 alpha primary antibodies

  • Description

    Rabbit polyclonal to HIF-1 alpha - ChIP Grade

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, WB, IP, IHC-Fr, ICC, ICC/IF, ChIPmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Guinea pig, Human, Xenopus laevis, Monkey
    Predicted to work with: Cow

  • Immunogen

    Fusion protein corresponding to Human HIF-1 alpha aa 432-528.

  • Positive control

    • ICC/IF: HeLa cells. IHC-P: Human kidney tissue. ChIP: HeLa cells. WB: Rat hypoxic nuclear lysate. IP: PC-3 cell lysate treated with cobalt chloride.

  • General notes

    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.

    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 ab2185 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
IHC-P 1/100.
WB 1/500 - 1/1000.
IP 1/1000.
IHC-Fr 1/10 - 1/2000.
ICC Use at an assay dependent concentration.
ICC/IF 1/10 - 1/2000.
EMSA 1/1 - 1/100.
ChIP Use at an assay dependent concentration.

25 µl / 15 millions cells

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

    see all

  • 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
    • 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

Images

  • ChIP - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    ChIP - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)This image is courtesy of a review submitted by Yolanda Cuevas, Hospital La Princesa.

    ChIP analysis of HIF-1-alpha genomic sequences from HeLa cells cultivated in normoxic (N) or hypoxic (Hx) conditions, using a HIF1-alpha polyclonal antibody (ab2185). For a negative control, IgG was used and the input as a positive control in the subsequent PCR. Primers for known target genes were used HIF1 alpha, A. EPO and B. VEGF.

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

    ICC/IF image of ab2185 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2185, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Western blot - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    Western blot - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    All lanes : Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

    Lane 1 : Rat nuclear extract lysate - normoxic
    Lane 2 : Rat nuclear extract lysate - hypoxic
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling HIF-1-alpha with ab2185.

  • Immunoprecipitation - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    Immunoprecipitation - Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
    All lanes : Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)

    Lane 1 : PC-3 cell lysates - untreated
    Lane 2 : PC-3 cell lysates - treated with 1µl cobalt chloride
    Lane 3 : PC-3 cell lysates - treated with 3µl cobalt chloride
    Lane 4 : PC-3 cell lysates - treated with 5µl cobalt chloride

References

This product has been referenced in:

  • Chen X  et al. Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling. J Cell Physiol 235:221-231 (2020). Read more (PubMed: 31187497) »
  • Liu FY  et al. TLR9 is essential for HMGB1-mediated post-myocardial infarction tissue repair through affecting apoptosis, cardiac healing, and angiogenesis. Cell Death Dis 10:480 (2019). Read more (PubMed: 31209243) »
See all 123 Publications for this product

Publishing research using ab2185? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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41-44 of 44 Abreviews or Q&A

Question

I am a student and I work on the ChIP IP I made a couple of Chip- Ip's with HIF 1 alpha, HIF 2 alpha and HIF 3 alpha. I collected between the process the supernatant and made western with the samples to have with the PCR a secound confirmation that the experiment worked. Now I started to clone the fragments. I made them blund end and purified them to make the ligation and transformation. I was not very sucessfull, because I had only religation and no inserts. I tried to find out how big my amount of sample is, but it is not enough to detecht by OD messerment orb with a DNA dot plot. My question now: Do you have any idea where I am loosing my DNA? The purification is exactly the same protocol you are using and the amount of cells is also fine. Three 15 c´m dishes fpr each condition. It would be great if you have some advices where to look for the problem.

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with your ChIP experiments. I am assuming that you are developing the assay to determine unknown HIF1 alpha, HIF2 and HIF3 binding sites. I would recommend that you perform experimental checks at each stage of this process. You are right to determine the success of the antibody by western analysis. Please can you tell me whether you were successful at detecting the protein in the recovered chromatin fraction. I would also recommend that you assay your recovered fraction using a quick PCR, amplifying a known HIF1 alpha binding region. This will enable you to determine whether your ChIP conditions are such that your proteins of interest are not only binding to the chromatin template but also present at "expected" regions. A reviewer who left favourable feedback used 25ul for 15 million of cells. It may be necessary to modify the volume of antibody that you are using. It may be useful to control for the cloning of the precipitated DNA (chromatin) fragments to determine whether it is your ChIP or final cloning that is to blame. I hope this information helps, please do not hesitate to contact us if you need any more advice or information or should you continue to have difficulty.

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Question

I order around two weeks ago two polyclonal antibodyes of HIF1 alpha (ab2185), when I went to used them instead of 100 microlitres, in the tube only Were 75 microlitres or less, this have hepened to me once time ago. Please contac with me to solve me the promblem, last time you send me a new sample. Tank you very much. Yours sincerely

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Answer

I'm terribly sorry about this. I will send to you straight away two new vials as soon as you provide me with the batch number and purchase order number or Abcam order number. My sincere apologies for the trouble,

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Question

Thank you for your ehlp. You were right. We purchased the vials via Biozol. Thank you vey much for offering us the refund. But as we urgently need this antibody, I would rather prefer to get another vial of the B2 or C lot, if available directly from you. Maybe there was something wrong with that shipment or the storage of that specific lot. If it is of any help, we could also send you two reference blots (one of the A lot and one of our B lot). There is a marked difference.

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Answer

As you urgently need this antibody, I suggest the best way to proceed is for you to place an order directly with Abcam. You may order online, via e-mail to orders@abcam.com, via fax to +44 1223 472038 or telephone +44 1223 472030. Please state when you place the order that you specifically wish to order batch B2. Currently, we have one vial of this lot in stock, the rest is all batch B1, so it is important for you to specify the lot. Please keep in touch and let me know how you get on with this batch. If it does not work as we say on the datasheet, we will refund you. Once you have the results with batch B2, we can decide on what action to take with regard to the vials of batch B1 that you purchased via Biozol. I'll keep my fingers crossed that batch B2 works in your research!

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Question

Researcher called to advise that he finds unsatisfactory results with batch B1 in WB. He has purchased two vials from this batch and both give the same results - lots of background bands in WB with a significantly fainter band at the expected size. Lot A gave a clean band at the right size. Is lot B1 from a different rabbit than lot A? What is the difference between lot B1 and lot B2? Same animal, different bleed?

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Answer

Abcam received the following response from Novus Biologicals: You are correct that we no longer have lot A available. Both lot A and B are from the same bunny. The only difference in lots A and B is the bleed. Lot A was actually from an earlier bleed. The only difference between lots B1 and B2 is the time that they were aliquotted. They are from the same bunny and bleed. We have not had complaints, either, about these lots. Therefore, I do not think that your customer will find any improvement using lot B2, opposed to lot B1. Hence, I suggest giving him a refund for one of the vials that he purchased. However, prior to giving him the refund, we require that he provide us with the detailed protocol that he used, along with a scan of his blot (including positive and negative controls). Thank you. + + + Please let me know how you wish to proceed. I assume that you have now contacted your purchasing agent to confirm if the vials were purchased via our German Distributor. I look forward to hearing from you.

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41-44 of 44 Abreviews or Q&A

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