Recombinant Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)
![Western blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)](https://www.abcam.com/ps/products/308/ab308637/Images/ab308637-1-anti-hif-1-alpha-hydroxyl-p564-antibody-epr27307-108-western-blot-untreated-hct116-human-colorectal-carcinoma-epithelial-cell-whole-cell-lysate-20-ug.jpg "Western blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR27307-108] to HIF-1 alpha (hydroxyl P564)
- Suitable for: ICC/IF, Dot blot, Flow Cyt (Intra), WB
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108]
See all HIF-1 alpha primary antibodies -
Description
Rabbit monoclonal [EPR27307-108] to HIF-1 alpha (hydroxyl P564) -
Host species
Rabbit -
Tested applications
Suitable for: ICC/IF, Dot blot, Flow Cyt (Intra), WBmore details
Unsuitable for: IHC-P -
Species reactivity
Reacts with: Human
Does not react with: Mouse, Rat -
Immunogen
This product was produced with the following immunogens:
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers. -
Positive control
- WB: MG-132 treated HCT116 whole cell lysate. MG-132 treated 293T cells transfected with a human HIFIA(WT) or human HIFIA P402G(mutant) expression vector containing a His-tag, whole cell lysate. ICC./IF: MG-132 treated HCT116 cells. Flow Cyt (Intra): MG-132 treated HCT116 cells.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 0.05% BSA, 40% Glycerol (glycerin, glycerine) -
Concentration information loading... -
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR27307-108 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab308637 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 |
1/50.
|
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| Dot blot |
1/1000.
|
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| Flow Cyt (Intra) |
1/50.
|
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| WB |
1/1000. Predicted molecular weight: 92 kDa.
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| Notes |
|---|
|
ICC/IF
1/50. |
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Dot blot
1/1000. |
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Flow Cyt (Intra)
1/50. |
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WB
1/1000. Predicted molecular weight: 92 kDa. |
Target
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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
modificationsIn 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
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Database links
- Entrez Gene: 3091 Human
- Omim: 603348 Human
- SwissProt: Q16665 Human
- Unigene: 597216 Human
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Alternative names
- ARNT interacting protein antibody
- ARNT-interacting protein antibody
- Basic helix loop helix PAS protein MOP1 antibody
see all
Images
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Western blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)All lanes : Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637) at 1/1000 dilution
Lane 1 : Untreated HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 2 : HCT116 treated with 10uM MG-132 for 4h whole cell lysate
Lane 3 : HIF1A knockout HCT116 whole cell lysate
Lane 4 : HIF1A knockout HCT116 treated with 10uM MG-132 for 4h whole cell lysate
Lane 5 : HCT116 treated with 0.5 mM DFO for 24 hour whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
In Western blot, Anti-HIF-1 alpha antibody (ab179483) as pan control staining at 1/1000 dilution.
The higher molecular weight band is the ubiquitylated forms of HIF1 alpha resulted from the hydroxylation of HIF1 alpha P564. VHLE3-dependent ubiquitination and proteasomal degradation described in PMID 10823831, PMID 11566883, PMID 12149254, PMID 21124777, PMID 10353251.
Cyclin D1(pT286)+cyclin D2(pT280)(ab230818) as control to prove MG-132 treatment is effective.
Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.
In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.
Exposure time: 147 seconds.
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![Western blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)](https://www.abcam.com/ps/products/308/ab308637/Images/ab308637-2-anti-hif-1-alpha-hydroxyl-p564-antibody-epr27307-108-western-blot-293t-cells-transfected-with-an-empty-vector-containing-a-his-tag-whole-cell-lysate-20-ug.jpg)
Western blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)All lanes : Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637) at 1/1000 dilution
Lane 1 : 293T cells transfected with an empty vector containing a His-tag whole cell lysate at 20 µg
Lane 2 : 293T cells transfected with an empty vector containing a His-tag, treated with 10 µM MG-132 for 4 hours whole cell lysate at 20 µg
Lane 3 : 293T cells transfected with a human HIFIA(WT) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 4 : 293T cells transfected with a human HIFIA(WT) expression vector containing a His-tag, treated with 10 µM MG-132 for 4 hours whole cell lysate at 20 µg
Lane 5 : 293T cells transfected with a human HIFIA P564G(mutant) expression vector containing a His-tag whole cell lysate at 50 µg
Lane 6 : 293T cells transfected with a human HIFIA P564G(mutant) expression vector containing a His-tag, treated with 10 µM MG-132 for 4 hours whole cell lysate at 50 µg
Lane 7 : 293T cells transfected with a human HIFIA P402G(mutant) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 8 : 293T cells transfected with a human HIFIA P402G(mutant) expression vector containing a His-tag, treated with 10 µM MG-132 for 4 hours whole cell lysate at 20 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
This antibody does not cross-react with human HIFIA (hydroxyl P402) .
In Western blot, Anti-HIF-1 alpha antibody (ab179483) as pan control staining at 1/1000 dilution.
In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.
Exposure time: 15 seconds.
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![Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)](https://www.abcam.com/ps/products/308/ab308637/Images/ab308637-3-anti-hif-1-alpha-hydroxyl-p564-antibody-epr27307-108-immunocytochemistry-hct-116.jpg)
Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labelling HIF-1 alpha (hydroxyl P564) with AB308637 at 1/50 dilution (10.32 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green).
Confocal image showing nuclear and weak cytoplasmic staining in HCT 116 cells after treatment with MG-132 (10 µM) for 4 hours and showing no staining in untreated HCT 116 cells.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (Red). The nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 ug/ml).
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![Flow Cytometry (Intracellular) - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)](https://www.abcam.com/ps/products/308/ab308637/Images/ab308637-4-anti-hif-1-alpha-hydroxyl-p564-antibody-epr27307-108-flow-cytometry-hct-116.jpg)
Flow Cytometry (Intracellular) - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT-116 (human colorectal carcinoma epithelial cell, Red) treated with 10 µM MG-132 for 4h / Untreated HCT-116 (Green) cells labelling HIF-1 alpha (hydroxyl P564) with AB308637 at 1/50 dilution (1 ug) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.
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![Dot Blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)](https://www.abcam.com/ps/products/308/ab308637/Images/ab308637-5-anti-hif-1-alpha-hydroxyl-p564-antibody-epr27307-108-dot-blot-human-hypoxia-inducible-factor-1-alphapeptide.jpg)
Dot Blot - Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)Dot blot analysis of HIF-1 alpha (hydroxyl P564) using AB308637 at 1:1000 (0.516 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.
Lane 1: Human Hypoxia-inducible factor 1-alpha peptide
Lane 2: Human Hypoxia-inducible factor 1-alpha(Hydroxyl-P402) peptide
Lane 3: Human Hypoxia-inducible factor 1-alpha (Hydroxyl-P564) peptideExposure time: 15 seconds.
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
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![Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)](https://www.abcam.com/ps/products/308/ab308637/Images/ab308637-506178-BenefitsofRecombinantantibodies.jpg)
Anti-HIF-1 alpha (hydroxyl P564) antibody [EPR27307-108] (ab308637)
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (0)
ab308637 has not yet been referenced specifically in any publications.