Product nameAnti-HIF-2-alpha antibody - ChIP Grade
See all HIF-2-alpha primary antibodies
DescriptionRabbit polyclonal to HIF-2-alpha - ChIP Grade
SpecificitySpecific for HIF-2-alpha / EPAS 1. Does not cross react with HIF-1-alpha.
Tested applicationsSuitable for: Flow Cyt, IHC-Fr, ChIP, IP, ICC/IF, IHC-P, WBmore details
Species reactivityReacts with: Mouse, Rat, Human
- CoCl2-treated Cos7 nuclear extract or hypoxic/normoxic PC12 nuclear extracts. Under normoxic conditions HIF-2 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-2 translocate to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880).
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.
Storage bufferpH: 7.50
Preservative: 0.05% Sodium azide
Constituent: 99% PBS
Concentration information loading...
- Pathways and Processes
- Metabolic signaling pathways
- Nucleotide metabolism
- Molecular processes
- Mitochondrial transcription
ChIP Related Products
Our Abpromise guarantee covers the use of ab199 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration.|
|ChIP||Use at an assay dependent concentration. PubMed: 19897487|
|IP||Use at 5-1 µg/mg of lysate.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|EMSA||Use at an assay dependent concentration.|
|WB||1/200 - 1/1000. Detects a band of approximately 118 kDa (predicted molecular weight: 100 kDa). The following should be taken into consideration: sufficient hypoxia induction is necessary; use nuclear extracts for cleaner results; use positive/negative controls to see which band is up-regulated; HIF2a degrades rapidly (fast sample preparation, protease inhibitors!).|
FunctionTranscription factor involved in the induction of oxygen regulated genes. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Regulates the vascular endothelial growth factor (VEGF) expression and seems to be implicated in the development of blood vessels and the tubular system of lung. May also play a role in the formation of the endothelium that gives rise to the blood brain barrier. Potent activator of the Tie-2 tyrosine kinase expression. Activation seems to require recruitment of transcriptional coactivators such as CREBPB and probably EP300. Interaction with redox regulatory protein APEX seems to activate CTAD.
Tissue specificityExpressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.
Involvement in diseaseDefects in EPAS1 are the cause of erythrocytosis familial type 4 (ECYT4) [MIM:611783]. ECYT4 is an autosomal dominant disorder characterized by increased serum red blood cell mass, elevated hemoglobin concentration and hematocrit, and normal platelet and leukocyte counts.
Sequence similaritiesContains 1 basic helix-loop-helix (bHLH) domain.
Contains 1 PAC (PAS-associated C-terminal) domain.
Contains 2 PAS (PER-ARNT-SIM) domains.
modificationsIn normoxia, is probably hydroxylated on Pro-405 and Pro-531 by EGLN1/PHD1, EGLN2/PHD2 and/or EGLN3/PHD3. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
In normoxia, is hydroxylated on Asn-847 by HIF1AN thus probably abrogating interaction with CREBBP and EP300 and preventing transcriptional activation.
Phosphorylated on multiple sites in the CTAD.
The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
- Information by UniProt
- Basic helix loop helix PAS protein MOP2 antibody
- Basic-helix-loop-helix-PAS protein MOP2 antibody
- bHLHe73 antibody
Analysis on normoxic and hypoxic nuclear rat cell lysates.
ab199 staining human adrenal tissue sections by IHC-P. Sections were PFA fixed and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking in 10% serum for 10 minutes at 25°C. The primary anitbody was diluted 1/500 and incubated with the sample for 1 hour at 25°C. A biotinylated goat anti-rabbit antibody was used as the secondary.
HIF-2α levels in HIF-2α−/− and WT mouse kidneys.
HIF-2α−/− mice or their wild-type littermates were exposed to left ureteral obstruction (UO), which continued for 24 hours, and then was released. 2 days after release of UO or at the corresponding time point in the non-UO sham-operated mice, the left kidneys were harvested and subjected to immunoblot analyses of HIF-2α and co-detection of TBP as a loading control
Nuclear extracts were isolated from harvested whole kidneys using NE-PER Nuclear and Cytoplasmic Extraction Reagents supplemented with Complete Protease Inhibitor Cocktail Tablets. Nuclear protein fractions were electrophoresed on 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. Membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE). Membranes were then incubated with the same blocking solution containing rabbit polyclonal primary antibodies against HIF-2α (1:500, ab199). After washing, membranes were incubated at room temperature for 1 h in TBS/0.05% Tween 20 buffer with the IRDye800 secondary antibodies (1:10000) and then washed again in TBS/0.05% Tween 20 for 3 times. The blot was visualized using an Odyssey infrared imaging system. All values were normalized to a loading control TATA binding protein (TBP, 1:2000, ab818, Abcam) and expressed as fold increase relative to control.
ICC/IF image of ab199 stained HepG2 cells. The cells were 4% PFA 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 (ab199, 1µg/ml) 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.
All lanes : Anti-HIF-2-alpha antibody - ChIP Grade (ab199) at 1/500 dilution
Lane 1 : 20ug of whole cell lysate from PC12 cells.
Lane 2 : 20ug of whole cell lysate from PC12 cells exposed to hypoxic conditions (12 hours at 3% oxygen).
Lane 3 : 20ug of whole cell lysate from PC12 cells exposed to hypoxic conditions (12 hours at 3% oxygen) and incubated with antisense HIF2 alpha oligonucleotides 72 hours prior to treatment.
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 100 kDa
Observed band size: 96 kDa why is the actual band size different from the predicted?
The blots were probed with anti-alpha tubulin as a loading control.
This product has been referenced in:
- Serocki M et al. miRNAs regulate the HIF switch during hypoxia: a novel therapeutic target. Angiogenesis 21:183-202 (2018). WB ; Human . Read more (PubMed: 29383635) »
- Rodrigues P et al. NF-?B-Dependent Lymphoid Enhancer Co-option Promotes Renal Carcinoma Metastasis. Cancer Discov 8:850-865 (2018). ChIP . Read more (PubMed: 29875134) »