Recombinant Anti-HIF-1 alpha antibody [BL-124-3F7] (ab243860)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [BL-124-3F7] to HIF-1 alpha
- Suitable for: WB, IHC-P, ICC, ChIP-sequencing, IP
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-HIF-1 alpha antibody [BL-124-3F7]
See all HIF-1 alpha primary antibodies -
Description
Rabbit monoclonal [BL-124-3F7] to HIF-1 alpha -
Host species
Rabbit -
Tested applications
Suitable for: WB, IHC-P, ICC, ChIP-sequencing, IPmore details -
Species reactivity
Reacts with: Human -
Immunogen
Recombinant fragment corresponding to Human HIF-1 alpha aa 433-826. NP_001521.1 and Gene ID 3091.
Database link: Q16665 -
Positive control
- ChIPseq: CoCl2 treated Hep-G2 cell lysate. WB: CoCl2 treated Hep-G2 and HeLa cell lysates. IHC-P: Human prostate carcinoma tissue. IP: CoCl2 treated Hep-G2 and HeLa cell lysates. ICC: CoCl2 treated HepG2 cells.
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General notes
This product is sold under License from Bethyl Laboratories, Inc.
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: 7.8
Preservative: 0.09% Sodium azide
Constituents: 98% Borate buffered saline, 0.1% BSA -
Concentration information loading...
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Purification notes
Recombinant antibody was purified from cell culture supernatant. -
Clonality
Monoclonal -
Clone number
BL-124-3F7 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab243860 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 |
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WB |
1/1000.
Ensure cell lysis occurs quickly if removed from hypoxia. Ultrasonic lysis is recommended to enrich more nuclear lysate. Positive Control: Hypoxic samples such as HeLa-DFO treated whole cell lysate ab116322. For a stronger signal, HeLa-DFO treated nuclear extracts are recommended ab180880. The cell fractionation kit can also be purchased separately ab109719. Negative Control: Most normal tissues or cells, other than kidney or heart. The observed band size of HIF-1 alpha is not exactly as predicted due to the different forms of HIF-1 alpha below: |
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IHC-P |
1/100 - 1/1000. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
Antigen retrieval with citrate buffer pH 6.0 for 20 minutes using a pressure cooker is recommended. |
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ICC |
1/100 - 1/1000.
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ChIP-sequencing |
Use a concentration of 10 - 30 µl/chromatin.
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IP |
Use at an assay dependent concentration.
Use 10µl/0.5mg lysate. |
Notes |
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WB
1/1000. Ensure cell lysis occurs quickly if removed from hypoxia. Ultrasonic lysis is recommended to enrich more nuclear lysate. Positive Control: Hypoxic samples such as HeLa-DFO treated whole cell lysate ab116322. For a stronger signal, HeLa-DFO treated nuclear extracts are recommended ab180880. The cell fractionation kit can also be purchased separately ab109719. Negative Control: Most normal tissues or cells, other than kidney or heart. The observed band size of HIF-1 alpha is not exactly as predicted due to the different forms of HIF-1 alpha below: |
IHC-P
1/100 - 1/1000. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. Antigen retrieval with citrate buffer pH 6.0 for 20 minutes using a pressure cooker is recommended. |
ICC
1/100 - 1/1000. |
ChIP-sequencing
Use a concentration of 10 - 30 µl/chromatin. |
IP
Use at an assay dependent concentration. Use 10µl/0.5mg lysate. |
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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Localization of human HIF1-alpha binding sites in immunoprecipitates from CoCl2 treated Hep-G2 lysate by ChIP-Seq using ab243860
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All lanes : Anti-HIF-1 alpha antibody [BL-124-3F7] (ab243860)
Lane 1 : 200M CoCl2 treated Hep-G2 cell lysate
Lane 2 : Mock treated Hep-G2 cell lysate
Lane 3 : 200M CoCl2 treated HeLa cell lysate
Lane 4 : Mock treated HeLa cell lysate
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution
Exposure time: 10 seconds -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [BL-124-3F7] (ab243860)Formalin-fixed, paraffin-embedded human prostate carcinoma tissue stained for HIF-1 alpha using ab243860 at 1/100 dilution in immunohistochemical analysis. A HRP-conjugated goat anti-rabbit IgG was used as the secondary. DAB staining.
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HIF-1 alpha was immunoprecipitated from 1mg CoCl2 and mock treated HepG2 and HeLa whole cell lysates with ab243860 at 20µg/mL lysate.
All lanes : Anti-HIF-1 alpha antibody [BL-124-3F7] (ab243860) at 1/1000 dilution
Lane 1 : IP using ab243860 in 200µM CoCl2 treated HepG2 cell lysate
Lane 2 : IP using ab243860 in mock treated HepG2 cell lysate
Lane 3 : IP using ab243860 in 200µM CoCl2 treated HeLa cell lysate
Lane 4 : IP using ab243860 in mock treated HeLa cell lysate
Lane 5 : IP using Control IgG in 200µM CoCl2 treated HepG2 cell lysate
Exposure time: 30 seconds -
Formalin-fixed, paraffin-embedded HepG2 (human liver hepatocellular carcinoma cell line) cells labeling HIF-1 alpha using ab243860 at 1/100 dilution in ICC analysis. A HRP-conjugated goat-anti rabbit IgG was used as the secondary. DAB staining.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [BL-124-3F7] (ab243860)Formalin-fixed, paraffin-embedded human renal cell carcinoma tissue stained for HIF-1 alpha using ab243860 at 1/100 dilution in immunohistochemical analysis. A HRP-conjugated goat anti-rabbit IgG was used as the secondary. DAB staining.
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)
ab243860 has been referenced in 2 publications.
- Xu K et al. HIF-1a regulates cellular metabolism, and Imatinib resistance by targeting phosphogluconate dehydrogenase in gastrointestinal stromal tumors. Cell Death Dis 11:586 (2020). PubMed: 32719331
- Yang K et al. Curcumin and Glu-GNPs Induce Radiosensitivity against Breast Cancer Stem-Like Cells. Biomed Res Int 2020:3189217 (2020). PubMed: 33457406