Overview

  • Product name

    Human HIF-1 alpha ELISA Kit, Fluorescent
    See all HIF-1 alpha kits
  • Detection method

    Fluorescent
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    DFO HeLa 5 4.3%
    Inter-assay
    Sample n Mean SD CV%
    DFO HeLa 3 7%
  • Sample type

    Cell culture extracts
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    12 pg/ml
  • Range

    0.03 ng/ml - 120 ng/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture media 112 94% - 129%
    Fetal Bovine Serum 109 102% - 120%
    Bovine Serum Albumin 97 93% - 106%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat
  • Product overview

    HIF-1 alpha in vitro CatchPoint® SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of HIF1a protein in human cell extracts.


    This CatchPoint SimpleStep ELISA kit has been optimized for Molecular Devices Microplate Readers. Click here for a list of recommended Microplate Readers.
    If using a Molecular Devices’ plate reader supported by SoftMax® Pro software, a preconfigured protocol for these CatchPoint SimpleStep ELISA Kits is available with all the protocol and analysis settings at www.softmaxpro.org.


    The CatchPoint® 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. CatchPoint® HRP Development Solution containing the Stoplight Red Substrate is added. During incubation, the substrate is catalyzed by HRP generating a fluorescent product. Signal is generated proportionally to the amount of bound analyte and the intensity is measured in a fluorescence plater reader at 530/570/590 nm Excitation/Cutoff/Emission.


     

  • Notes

    Hypoxia-inducible factor 1-alpha (HIF1 alpha) is a constitutively expressed transcription factor that is degraded under normal oxygen tensions but stabilized when oxygen is limiting (hypoxia). Under hypoxic conditions, stabilized HIF1 alpha translocates to the nucleus and promotes the transcription of a host of genes that enable the cell to adapt to the lack of oxygen. Aspects of the HIF1 alpha mediated hypoxic response include promotion of angiogenesis and the switch from aerobic respiration to anaerobic glycolysis. Many of the HIF1 alpha responsive genes encode proteins that promote glycolysis and/or inhibit oxidative phosphorylation (known as the Warburg effect). An exciting and developing area of current cancer research is examining how HIF-mediated metabolic reprogramming promotes tumor growth and survival.

    In most cases, HIF1 alpha will need to be stabilized to be measured (steady state levels of HIF1 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 HIF1 alpha in normoxia. By disrupting the enzymes that degrade HIF1 alpha, DFO increases the abundance of HIF1 alpha protein.

  • Tested applications

    Suitable for: Sandwich 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
    100X Stoplight Red Substrate 1 x 120µl
    10X Human HIF1a Capture Antibody 1 x 600µl
    10X Human HIF1a Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    500X Hydrogen Peroxide (H2O2, 3%) 1 x 50µl
    50X Cell Extraction Enhancer Solution 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml
    Antibody Diluent 5B 1 x 6ml
    HIF1a Human Lyophilized Protein 2 vials
    Plate Seals 1 unit
    Sample Diluent NS (ab193972) 1 x 50ml
    SimpleStep Pre-Coated Black 96-Well Microplate 1 unit
    Stoplight Red Substrate Buffer 1 x 12ml
  • Research areas

  • 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
    modifications

    In 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
  • Alternative names

    • ARNT interacting protein
    • ARNT-interacting protein
    • Basic helix loop helix PAS protein MOP1
    • Basic-helix-loop-helix-PAS protein MOP1
    • bHLHe78
    • Class E basic helix-loop-helix protein 78
    • HIF 1A
    • HIF 1alpha
    • HIF-1-alpha
    • HIF-1alpha
    • HIF-alpha
    • HIF1
    • HIF1 A
    • HIF1 Alpha
    • HIF1-alpha
    • HIF1A
    • HIF1A_HUMAN
    • hifla
    • Hypoxia inducible factor 1 alpha
    • Hypoxia inducible factor 1 alpha isoform I.3
    • Hypoxia inducible factor 1 alpha subunit
    • Hypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor
    • Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor)
    • Hypoxia inducible factor1alpha
    • Hypoxia-inducible factor 1-alpha
    • Member of PAS protein 1
    • Member of PAS superfamily 1
    • Member of the PAS Superfamily 1
    • MOP 1
    • MOP1
    • PAS domain-containing protein 8
    • PASD 8
    • PASD8
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab229433 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
Sandwich 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.

     

  • The HIF1a standard curve was prepared as described in Section 10. Raw data generated on SpectraMax M4 Multi-Mode Microplate Reader is shown in the table. Background-subtracted data values (mean +/- SD) are graphed.

  • HeLa cells were cultured in 96-well tissue culture plates and were either untreated or exposed to varying dose of DFO for 24 hours. Cells were extracted directly in the culture plate by overlaying culture media with Extraction Buffer PTR (with Extraction Enhancer) such that the final concentration was 1X Extraction Buffer. Extracts were applied to the HIF1 alpha ELISA. Raw data with standard deviation is plotted from triplicate measurements.

  • Background subtracted OD450 nm data from three loading concentrations are shown. In the HeLa cell line, DFO treatment is required to detect HIF1 alpha protein.

  • Background subtracted data from duplicate measurements are plotted. To induce HIF1 alpha protein levels, HeLa cells were treated with 500 µM Deferoxamine (DFO) for 24 hours.

Protocols

References

ab229433 has not yet been referenced specifically in any publications.

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