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 it.

    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
  • 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

  • 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.

  • 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.

  • 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) analysis of Human kidney tissue sections labeling HIF-1-alpha with 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:
  • Xu Q  et al. Angiogenesis for tumor vascular normalization of Endostar on hepatoma 22 tumor-bearing mice is involved in the immune response. Oncol Lett 15:3437-3446 (2018). Read more (PubMed: 29467868) »
  • Simone L  et al. Aquaporin-1 inhibition reduces metastatic formation in a mouse model of melanoma. J Cell Mol Med 22:904-912 (2018). Mouse . Read more (PubMed: 29044946) »
See all 96 Publications for this product

Customer reviews and Q&As

1-10 of 44 Abreviews or Q&A

Answer

Thank you for your enquiry regarding ab2185 - Anti-HIF1 alpha antibody - ChIP Grade.

I can confirm that ab2185 is a polyclonal antibody and not affinity purified; it is whole antiserum. As you may be aware there is usually batch-to-batch variations for polyclonals (vs monoclonals) depending on the immune response of the host animals (rabbit). I would advise you to use the recommended cell numbers indicated on the product datasheet which is regularly updated with the most up-to-date information.

I hope this will be useful for you. Should you still have any question with this antibody, then please do not hesitate to contact our Technical Department again.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK293)
Permeabilization
Yes - 0.1% Triton X100
Specification
HEK293
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 18 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Notothenia coriiceps Cell lysate - other (heart)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Treatment
CTmax
Specification
heart
Blocking step
Milk as blocking agent for 10 hour(s) and 0 minute(s) · Concentration: 0.1% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jan 13 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Chaenocephalus aceratus Cell lysate - other (heart)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Treatment
CTmax
Specification
heart
Blocking step
BSA as blocking agent for 10 hour(s) and 0 minute(s) · Concentration: 0.1% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jan 13 2017

Question
Answer

The protein HIF-1alpha does translocate to nucleus under hypoxic conditions, therefore it will be worth to try nuclear lysates as well.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (necrotic melanoma xenograft)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9
Permeabilization
No
Specification
necrotic melanoma xenograft
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 18 2015

Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample
Mouse Cultured Cells (Neuro2A- neuroblastoma)
Specification
Neuro2A- neuroblastoma
Permeabilization
Yes - 0.3% Triton X-100
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 04 2013

Answer

Thank you for contacting us.

ab2185 Immunogen sequence is more than 90% similar with Pig HIF1 alpha so this antibody is more likely to cross react.

http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20130405-144240-0769-61535367-pg

ab21685 is anti GRP78

GRP93 there is no protein with this name. Do you think it is GRP94?

ab119 would be suitable as anti VEGF. We do have other antibodies as well but they are yet to be tested in Pig. if you are interested in testing then you may be interested in Abtrial discount programme. Please click the link for more information.

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.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Striatum (Brain))
Loading amount
50 µg
Specification
Striatum (Brain)
Gel Running Conditions
Reduced Denaturing (8%)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 14 2013

Question
Answer

Thank you for your reply. The immunogen shows 89% homology with the immunogen. We are not able to obtain consistent results with rat samples therefore are not able to guarantee that it will give positive results. However, customers have shown that this antibody successfully detects rat HIF1 alpha. Please see the Abreviews on the datasheet for more information. We have 3 other Hif1 alpha antibodies tested in rat that would be more suitable: ab65979 Anti-HIF1 alpha antibody - Carboxyterminal end https://www.abcam.com/ab65979 ab463 Anti-HIF1 alpha antibody [H1alpha67-sup] https://www.abcam.com/ab463 ab2185 Anti-HIF1 alpha antibody - ChIP Grade https://www.abcam.com/ab2185 Just let me know if you have further questions.

Read More

1-10 of 44 Abreviews or Q&A

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