Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
- Datasheet
- Specific References (84)
- Protocols
Overview
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Product nameAnti-HIF-1 alpha antibody - ChIP Grade
See all HIF-1 alpha primary antibodies -
DescriptionRabbit polyclonal to HIF-1 alpha - ChIP Grade
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Host speciesRabbit
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Tested applicationsSuitable for: IHC-P, WB, IP, IHC-Fr, ICC, ICC/IF, ChIPmore details
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Species reactivityReacts with: Mouse, Rat, Guinea pig, Human, Xenopus laevis, Monkey
Predicted to work with: Cow
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Immunogen
Fusion protein corresponding to Human HIF-1 alpha aa 432-528.
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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
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FormLiquid
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
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Storage bufferPreservative: 0.05% Sodium azide
Constituents: Tris glycine, 0.87% Sodium chloride -
Concentration information loading... -
PurityImmunogen affinity purified
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ClonalityPolyclonal
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IsotypeIgG
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Research areas
Associated products
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Assay kits
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ChIP Related Products
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Compatible Secondaries
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Recombinant Protein
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Related Products
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
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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.
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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.
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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).
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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 localizationCytoplasm. 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: 281814 Cow
- Entrez Gene: 3091 Human
- Entrez Gene: 15251 Mouse
- Entrez Gene: 29560 Rat
- Entrez Gene: 100337573 Xenopus laevis
- Entrez Gene: 380141 Xenopus laevis
- Omim: 603348 Human
- SwissProt: Q16665 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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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.
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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.
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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.
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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 -
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
Protocols
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) »
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