Mouse, Rat, Human Predicted to work with:
Chimpanzee, Macaque monkey, Chinese hamster, Orangutan
Synthetic peptide corresponding to Human HIF-2-alpha aa 350-450 conjugated to Keyhole Limpet Haemocyanin (KLH). (Peptide available as ab133169)
WB: Human placenta and heart tissue lysates and A549, HeLa, NIH3T3 and PC12 whole cell lysates.
IHC: Human colon tissue.
ICC/IF: HeLa cells (untreated and treated with Deferoxamine).
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
pH: 7.40 Preservative: 0.02% Sodium azide Constituent: PBS Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 96 kDa).
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Use a concentration of 10 µg/ml.
Transcription 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.
Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.
Involvement in disease
Defects 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.
In 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.
ab109616 staining Hif-2-alpha in HeLa cells. The cells were incubated with 1mM Deferoxamine for 24 hours (Treated) or solvent-only for control purposes (Solvent only). Cells were fixed with 4% formaldehyde (10min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab109616 at 10µg/ml and ab195889 at 1µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
IHC image of HIF-2alpha staining in a section of formalin-fixed paraffin-embedded normal human colon*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 for 20mins. The section was then incubated with ab109616 at 1μg/ml, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Western blot - Anti-HIF-2-alpha antibody (ab109616)
All lanes : Anti-HIF-2-alpha antibody (ab109616) at 1 µg/ml (Milk blocking - 3%)
Lane 1 : Human placenta tissue lysate - total protein (ab29745) Lane 2 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate Lane 3 : Human heart tissue lysate - total protein (ab29431) Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lysates/proteins at 20 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
The predicted molecular weight of HIF2 alpha is 96 kDa (SwissProt), however we expect to observe a banding pattern around 120 kDa. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.