Anti-HIF-1 alpha antibody - ChIP Grade (ab2185)
- Datasheet
- References (123)
- Protocols
Overview
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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, GSA, IHC-Fr, ICC, ICC/IF, ChIPmore details -
Species reactivity
Reacts 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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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.
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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 the English version and here to download the Mandarin.
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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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
Preservative: 0.05% Sodium azide
Constituent: PBS -
Concentration information loading... -
Purity
Immunogen affinity purified -
Clonality
Polyclonal -
Isotype
IgG -
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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Isotype control
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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. | |
| GSA | Use at an assay dependent dilution. | |
| 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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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: 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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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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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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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 -
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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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:
- Chen X et al. Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling. J Cell Physiol 235:221-231 (2020). Read more (PubMed: 31187497) »
- Liu FY et al. TLR9 is essential for HMGB1-mediated post-myocardial infarction tissue repair through affecting apoptosis, cardiac healing, and angiogenesis. Cell Death Dis 10:480 (2019). Read more (PubMed: 31209243) »
