Key features and details
- One-wash 90 minute protocol
- Sensitivity: 19 pg/ml
- Range: 31.3 pg/ml - 2000 pg/ml
- Sample type: Cell culture extracts
- Detection method: Colorimetric
- Assay type: Sandwich (quantitative)
- Reacts with: Human
Product nameHuman HIF-2-alpha ELISA Kit
See all HIF-2-alpha kits
Intra-assay Sample n Mean SD CV% HeLa extract 5 2.6% Inter-assay Sample n Mean SD CV% HeLa extract 3 4%
Sample typeCell culture extracts
Assay typeSandwich (quantitative)
Range31.3 pg/ml - 2000 pg/ml
Sample specific recovery Sample type Average % Range Cell culture extracts 114 112% - 116%
Assay time1h 30m
Assay durationOne step assay
Species reactivityReacts with: Human
Human HIF-2-alpha ELISA Kit (ab227898) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of HIF-2-alpha protein in cell culture extracts. It uses our proprietary SimpleStep ELISA® technology. Quantitate Human HIF-2-alpha with 19 pg/ml sensitivity.
SimpleStep ELISA® technology employs capture antibodies conjugated to an affinity tag that is recognized by the monoclonal antibody used to coat our SimpleStep ELISA® plates. This approach to sandwich ELISA allows the formation of the antibody-analyte sandwich complex in a single step, significantly reducing assay time. See the SimpleStep ELISA® protocol summary in the image section for further details. Our SimpleStep ELISA® technology provides several benefits:
- Single-wash protocol reduces assay time to 90 minutes or less
- High sensitivity, specificity and reproducibility from superior antibodies
- Fully validated in biological samples
- 96-wells plate breakable into 12 x 8 wells strips
A 384-well SimpleStep ELISA® microplate (ab203359) is available to use as an alternative to the 96-well microplate provided with SimpleStep ELISA® kits.
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
PlatformPre-coated microplate (12 x 8 well strips)
Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 10X Human HIF-2-alpha Capture Antibody 1 x 600µl 10X Human HIF-2-alpha Detector Antibody 1 x 600µl 10X Wash Buffer PT (ab206977) 1 x 20ml 50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml 5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml Antibody Diluent CPR 1 x 6ml Human HIF-2-alpha Lyophilized Recombinant Protein 2 vials Plate Seals 1 unit Sample Diluent NS (ab193972) 1 x 12ml SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit Stop Solution 1 x 12ml TMB Development Solution 1 x 12ml
- Pathways and Processes
- Metabolic signaling pathways
- Nucleotide metabolism
- Molecular processes
- Mitochondrial transcription
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
- Basic-helix-loop-helix-PAS protein MOP2
SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
Background-subtracted data values (mean +/- SD) are graphed.
HELA cells were untreated or treated with 250 µM DFO for 24 hrs, then collected and extracted according to sample preparation protocol (see section 11.1). The concentrations of HIF-2-alpha were measured in duplicate and interpolated from the HIF-2-alpha standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean HIF-2-alpha concentration was determined to be 520 pg/mL in DFO treated HELA cell extract and undetectable in untreated HELA cell extract.
HeLa cells were cultured in 96-well tissue culture plates and were either untreated (Vehicle Control) or exposed to varying doses of DFO for 24 hours (DFO treated). Cells were extracted directly in the culture plate by overlaying culture media with Cell Extraction Buffer PTR with Enhancer such that the final concentration was 1X Cell Extraction Buffer PTR. Extracts were applied to the HIF-2-alpha SimpleStep ELISA plate. Raw data and standard deviation is plotted from quadruplicate measurements.
To learn more about the advantages of recombinant antibodies see here.
ab227898 has not yet been referenced specifically in any publications.