Recombinant Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)
![Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)](https://www.abcam.com/ps/products/279/ab279654/Images/ab279654-481459-279654dhew6247westernblotwildtypehct116untreateddmogcontrolwildtypehct116treateddmog.jpg "Western blot - Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Mouse monoclonal [54/HIF-1a] to HIF-1 alpha
- Suitable for: ICC/IF, WB
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-HIF-1 alpha antibody [54/HIF-1a]
See all HIF-1 alpha primary antibodies -
Description
Mouse monoclonal [54/HIF-1a] to HIF-1 alpha -
Host species
Mouse -
Tested applications
Suitable for: ICC/IF, WBmore details
Unsuitable for: IHC-P -
Species reactivity
Reacts with: Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa treated with 1 mM DFO (Deferoxamine mesylate) for 24 hrs, whole cell lysate. HeLa treated with 0.5 mM CoCl2 for 6 hrs, whole cell lysate. ICC/IF: HeLa cells treated with 1 mM desferrioxamine for 24 hrs.
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General notes
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.
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
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
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading... -
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
54/HIF-1a -
Isotype
IgG1 -
Light chain type
kappa -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab279654 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 |
1/20.
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| WB |
1/2000. Predicted molecular weight: 92 kDa.
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| Notes |
|---|
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ICC/IF
1/20. |
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WB
1/2000. 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 antibody [54/HIF-1a] (ab279654)All lanes : Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654) at 1/2000 dilution
Lane 1 : Wild-type HCT 116 Untreated DMOG Control cell lysate
Lane 2 : Wild-type HCT 116 Treated DMOG (1 mM, 4 h) cell lysate
Lane 3 : HIF1A knockout HCT 116 Untreated DMOG Control cell lysate
Lane 4 : HIF1A knockout HCT 116 Treated DMOG (1 mM, 4 h) cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 92 kDaFalse colour image of Western blot: Anti-HIF-1 alpha antibody [54/HIF-1a] staining at 1/2000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279654 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in treated wild-type HCT 116 cell lysates with no signal observed at this size in HIF1A knockout cell line ab255394 (knockout cell lysate ab263860). To generate this image, wild-type and HIF1A knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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![Western blot - Anti-HIF-1 alpha antibody [54] (ab279654)](https://www.abcam.com/ps/products/279/ab279654/Images/ab279654-453350-anti-hif1-alpha-antibody-54-western-blot-untreated-hela.jpg)
Western blot - Anti-HIF-1 alpha antibody [54] (ab279654)All lanes : Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654) at 1/2000 dilution
Lane 1 : Untreated HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HeLa treated with 1 mM DFO (Deferoxamine mesylate) for 24 hours whole cell lysate
Lane 3 : Untreated HeLa whole cell lysate
Lane 4 : HeLa treated with 0.5 mM CoCl2 for 6 hours whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/50000 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.
The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID: 9159130).
Exposure time: 3 minutes.
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![Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)](https://www.abcam.com/ps/products/279/ab279654/Images/ab279654-1-anti-hif1-alpha-antibody-54-immunocytochemistry-hela.jpg)
Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling HIF-1 alpha with ab279654 at 1/20 dilution, followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green).
Confocal image showing nuclear staining in HeLa cells treated with 1 mM desferrioxamine for 24 hrs.
ab179513 anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Red). The nuclear counterstain was DAPI (Blue).
Secondary antibody only controls: Secondary antibodies are ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution and ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at 1/1000 dilution.
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![Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)](https://www.abcam.com/ps/products/279/ab279654/Images/ab279654-456285-benefitsofrecombinantantibodies.jpg)
Anti-HIF-1 alpha antibody [54/HIF-1a] (ab279654)
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
References (2)
ab279654 has been referenced in 2 publications.
- Meng F et al. LncRNA LINC00525 activates HIF-1a through miR-338-3p / UBE2Q1 / ß-catenin axis to regulate the Warburg effect in colorectal cancer. Bioengineered 13:2554-2567 (2022). PubMed: 35156520
- Zhang J et al. Combining immune checkpoint blockade with ATP-based immunogenic cell death amplifier for cancer chemo-immunotherapy. Acta Pharm Sin B 12:3694-3709 (2022). PubMed: 36176905