Anti-HIF1 alpha antibody [mgc3] (ab16066)
- Product nameAnti-HIF1 alpha antibody [mgc3]See all HIF1 alpha primary antibodies ...
- DescriptionMouse monoclonal [mgc3] to HIF1 alpha
- SpecificityThis antibody does not cross-react with ARNT or the related HIF2 alpha.
- Tested applicationsFlow Cyt, EMSA, Gel supershift assays, ICC/IF, IP, WB, IHC-P more details
- Species reactivityReacts with: Cow, Human, Pig
Predicted to work with: Non Human Primates
Synthetic peptide, corresponding to C terminal amino acids 530-826 of Human HIF-1 alpha.
- Positive controlCOS-7 cells treated with 1% oxygen for 4 hours to induce hypoxia.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPreservative: 0.05% Sodium Azide
Constituents: 1mg/ml BSA
- Concentration information loading...
- PurityProtein G purified
- Clonality Monoclonal
- Clone numbermgc3
Our Abpromise guarantee covers the use of ab16066 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Flow Cyt: Use 2µg for 106 cells.|
|EMSA||EMSA: Use at an assay dependent dilution. PubMed: 21193390|
|Gel supershift assays||GSA: Use at an assay dependent dilution. Only been successfully used with mouse HIF1 alpha.|
|ICC/IF||ICC/IF: Use at an assay dependent dilution.|
|IP||IP: Use at an assay dependent dilution.|
|WB||WB: 1/2000. Detects a band of approximately 116 kDa (predicted molecular weight: 93 kDa). 116 kDa band represents HIF-1 alpha after hypoxic induction.|
|IHC-P||IHC-P: 1/800. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. See Abreview for further information|
- FunctionFunctions 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 specificityExpressed 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 similaritiesContains 1 basic helix-loop-helix (bHLH) domain.
Contains 1 PAC (PAS-associated C-terminal) domain.
Contains 2 PAS (PER-ARNT-SIM) domains.
- DomainContains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
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 localizationCytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
- ARNT interacting protein antibodyARNT interacting protein antibodyARNT-interacting protein antibody
- Basic helix loop helix PAS protein MOP1 antibodyBasic-helix-loop-helix-PAS protein MOP1 antibodybHLHe78 antibodyClass E basic helix-loop-helix protein 78 antibodyHIF 1A antibodyHIF 1alpha antibodyHIF-1-alpha antibodyHIF1 A antibodyHIF1 Alpha antibodyHIF1 antibodyHIF1-alpha antibodyHIF1A antibodyHIF1A antibodyHIF1A_HUMAN antibodyHypoxia inducible factor 1 alpha antibodyHypoxia inducible factor 1 alpha antibodyHypoxia inducible factor 1 alpha isoform I.3 antibodyHypoxia inducible factor 1 alpha subunit antibodyHypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor antibodyHypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor antibodyHypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor) antibodyHypoxia inducible factor1alpha antibodyHypoxia-inducible factor 1-alpha antibodyMember of PAS protein 1 antibodyMember of PAS superfamily 1 antibodyMember of PAS superfamily 1 antibodyMember of the PAS Superfamily 1 antibodyMOP 1 antibodyMOP 1 antibodyMOP1 antibodyPAS domain-containing protein 8 antibodyPASD 8 antibodyPASD8 antibody
Anti-HIF1 alpha antibody [mgc3] images
Anti-HIF1 alpha antibody [mgc3] (ab16066) at 1/2000 dilution + Hela cell lysate
Predicted band size : 93 kDa
Observed band size : 116 kDa (why is the actual band size different from the predicted?)
Immunofluorescence staining of HIF-1 alpha using ab16066
ab16066 staining HIF1 alpha in Human small intestine (IBD) and tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde; antigen retrieval was by heat mediation with an EDTA buffer (pH 9.0). Samples were incubated with primary antibody (1/800 in diluent + background reducers) for 20 minutess at 25°C. An undiluted Goat polymer was used as the secondary antibody.
Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing HIF-1 alpha ab16066 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.
Overlay histogram showing HeLa cells stained with ab16066 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16066, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Immunofluorescence analysis of Human fibroblasts, staining HIF1 alpha (green) with ab16066.
Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 1 hour at 24°C. Samples were incubated with primary antibody (1/500 in 0.3% triton X-100 + 1% BSA) for 1 hour 30 minutes at 24°C. An AlexaFluor®488-conjugated goat anti-mouse polyclonal IgG (1/2000) was used as the secondary antibody.
References for Anti-HIF1 alpha antibody [mgc3] (ab16066)
This product has been referenced in:
- Guadall A et al. Fibulin-5 Is Up-regulated by Hypoxia in Endothelial Cells through a Hypoxia-inducible Factor-1 (HIF-1α)-dependent Mechanism. J Biol Chem 286:7093-103 (2011). EMSA ; Human . Read more (PubMed: 21193390) »
- Kristiansen G et al. Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype. Br J Cancer 102:1736-45 (2010). IHC-P ; Human . Read more (PubMed: 20531416) »