Overview

  • Product name

    Human HIF-2-alpha ELISA Kit
    See all HIF-2-alpha kits
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    HeLa extract 5 2.6%
    Inter-assay
    Sample n Mean SD CV%
    HeLa extract 3 4%
  • Sample type

    Cell culture extracts
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    19 pg/ml
  • Range

    31.3 pg/ml - 2000 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture extracts 114 112% - 116%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    HIF-2-alpha in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of HIF-2-alpha protein in human cell and tissue extracts.


    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.


    Hypoxia-inducible factor 2-alpha (HIF-2-alpha) is a constitutively expressed transcription factor that is degraded under normal oxygen tensions but stabilized when oxygen is limiting (hypoxia).   HIF-2-alpha is similar to HIF-1-alpha. HIF-1-alpha is most active during the hypoxic trigger (2-24 hrs), while HIF-2-alpha is more active in the 2-3 days following the hypoxic trigger. In most cases, HIF-2-alpha will need to be stabilized to be measured (steady state levels of HIF-2-alpha in non-hypoxic environments is exceeding low in most cell lines). This can be achieved by (a) creating a hypoxic environment (e.g. using a hypoxia chamber) or (b) by using chemical treatments that mimic hypoxia (e.g. cobalt chloride or deferoxamine). The sample data in this assay protocol was generated using deferoxamine (DFO). DFO is an iron chelator and disrupts the function the prolyl hydroxylases that degrade HIF-2-alpha in normoxia. By disrupting the enzymes that degrade HIF-2-alpha, DFO increases the abundance of HIF-2-alpha protein.

  • Tested applications

    Suitable for: ELISAmore details
  • Platform

    Pre-coated microplate (12 x 8 well strips)

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    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
    10X Human HIF-2-alpha Capture Antibody 1 x 600µl
    10X Human HIF-2-alpha Detector Antibody 1 x 600µl
    Human HIF-2-alpha Lyophilized Recombinant Protein 1 x 2 vials
    Plate Seals 1 unit
    Sample Diluent NS 1 x 12ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Development Solution 1 x 12ml
  • Research areas

  • Function

    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.
  • Tissue specificity

    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.
  • 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.
  • Post-translational
    modifications

    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.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Alternative names

    • Basic helix loop helix PAS protein MOP2
    • Basic-helix-loop-helix-PAS protein MOP2
    • bHLHe73
    • Class E basic helix-loop-helix protein 73
    • ECYT4
    • Endothelial PAS domain containing protein 1
    • Endothelial pas domain protein 1
    • Endothelial PAS domain-containing protein 1
    • EPAS 1
    • EPAS-1
    • EPAS1
    • EPAS1_HUMAN
    • HIF 1 alpha like factor
    • HIF 2 alpha
    • HIF-1-alpha-like factor
    • HIF-2-alpha
    • HIF2-alpha
    • HIF2A
    • HLF
    • Hypoxia inducible factor 2 alpha
    • Hypoxia inducible factor 2 alpha subunit
    • Hypoxia-inducible factor 2-alpha
    • Member of PAS protein 2
    • Member of pas superfamily 2
    • MOP 2
    • MOP2
    • PAS domain-containing protein 2
    • PASD2
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab227898 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
ELISA Use at an assay dependent concentration.

Images

  • 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.

Protocols

References

ab227898 has not yet been referenced specifically in any publications.

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