Overview

  • Product name

    Human Hif1a + BNIP3 Hypoxia In Cell ELISA Kit (IR)
  • Detection method

    IR
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    ab129733 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure HIF1 alpha and BNIP3 protein levels in cultured cells. Cells are fixed in a microplate and targets of interest are detected with highly specific, well-characterized antibodies. Relative protein levels are quantified using IRDye®-labeled Secondary Antibodies and IR imaging using a LI-COR® Odyssey® or Aerius® system.

     

    Hypoxia and the cellular response to hypoxic environment is a central topic in studies of metabolism, cancer progression and development and stem cells. A key player is the transcription factor HIF1 alpha (hypoxia inducible factor 1 alpha) which is stabilized at the protein level in response to decreased oxygen tension. HIF1 alpha then promotes transcription of a number of factors that alters cellular physiology. This Hypoxia ICE assay kit provides duplexed measurements of the transcription factor HIF1 alpha and the HIF1A responsive protein BNIP3.

     

    Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    1000X IRDye-labeled Secondary Antibodies 1 x 24µl
    100X BNIP3 Primary Antibody (Mouse) 1 x 120µl
    100X HIF1alpha Primary Antibody (Rabbit) 1 x 120µl
    100X Triton X-100 1 x 0.5ml
    10X Blocking Solution 1 x 10ml
    10X Phosphate Buffered Saline 1 x 100ml
    400X Tween-20 1 x 2ml
    1X Janus Green Stain 1 x 17ml
  • Research areas

  • Relevance

    The gene encodes the alpha subunit of transcription factor hypoxia-inducible factor-1 (HIF-1), which is a heterodimer composed of an alpha and a beta subunit. HIF-1 functions as a master regulator of cellular and systemic homeostatic response to hypoxia by activating transcription of many genes, including those involved in energy metabolism, angiogenesis, apoptosis, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. HIF-1 thus plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Alternatively spliced transcript variants encoding different isoforms have been identified for this gene. The gene is a member of the BCL2/adenovirus E1B 19 kd-interacting protein (BNIP) family. It interacts with the E1B 19 kDa protein, which protects cells from virally-induced cell death. The encoded protein also interacts with E1B 19 kDa-like sequences of BCL2, another apoptotic protector. This protein contains a BH3 domain and a transmembrane domain, which have been associated with pro-apoptotic function. The dimeric mitochondrial protein encoded by this gene is known to induce apoptosis, even in the presence of BCL2.
  • Cellular localization

    HIF1a: Cytoplasmic and Nuclear. Note: Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia. BNIP3: Mitochondrion. Mitochondrion membrane; Single-pass membrane protein. Note: Coexpression with the EIB 19-kDa protein results in a shift in NIP3 localization pattern to the nuclear envelope. Colocalizes with ACAA2 in the mitochondria.
  • Alternative names

    • ARNT interacting protein
    • ARNT-interacting protein
    • Basic helix loop helix PAS protein MOP1
    • Basic-helix-loop-helix-PAS protein MOP1
    • BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
    • BCL2/adenovirus E1B 19kDa interacting protein 3
    • bHLHe78
    • Class E basic helix-loop-helix protein 78
    • HIF 1A
    • HIF 1alpha
    • HIF-1-alpha
    • HIF-1alpha
    • HIF1
    • HIF1 A
    • HIF1 Alpha
    • HIF1-ALPHA
    • HIF1A
    • HIF1A_HUMAN
    • 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 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
    • NIP3
    • PAS domain-containing protein 8
    • PASD 8
    • PASD8
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab129733 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
In-Cell ELISA Use at an assay dependent concentration.

Images

  • Coefficient of variation for the experiment described in Figure 1.
  • Antibody specificity demonstrated by Western Blot. Primary antibodies used in this assay kit were validated by Western Blot using HeLa cell lysates that had been treated with a dose titration of DFO as indicated. (A) The HIF1 alpha band (indicated by arrow) is absent in untreated cells and induced by DFO. (B) Similarly, BNIP3 levels are increased by DFO treatment in a dose-dependent manner.
  • Antibody specificity demonstrated by immunocytochemistry. Primary antibodies used in this assay kit were validated by staining HeLa cells +/- treatment with 1mM DFO (24h) and imaged by fluorescent microscopy. BNIP3 staining is nearly undetectable in untreated HeLa cells but is induced by DFO treatment. BNIP3 appears to has a mitochondrial staining pattern in the DFO-treated samples.
  • Antibody specificity demonstrated by immunocytochemistry. Primary antibodies used in this assay kit were validated by staining HeLa cells +/- treatment with 1mM DFO (24h) and imaged by fluorescent microscopy. HIF1 alpha staining is absent in untreated cells and induced by DFO treatment. HIF1 alpha localizes to the nucleus (as seen by co-localization with the DNA stain DAPI) as expected.
  • Sample experiment using ab129733 on HeLa cells treated with a titration of DFO. HeLa cells were seeded to an amine coated 96-well microplate and the following day treated with a titration of DFO. After 24h of DFO exposure, the cells were fixed and stained as described in the protocol and the normalized data is presented here +/- SD (as described in the protocol and data analysis sections). BNIP3 levels are increased with DFO concentrations ≥10µM.
  • Sample experiment using ab129733 on HeLa cells treated with a titration of DFO. HeLa cells were seeded to an amine coated 96-well microplate and the following day treated with a titration of DFO. After 24h of DFO exposure, the cells were fixed and stained as described in the protocol and the normalized data is presented here +/- SD (as described in the protocol and data analysis sections). HIF1 alpha results show DFO concentrations ≥10µM induce HIF1A protein levels in a dose dependent manner.

Protocols

References

ab129733 has not yet been referenced specifically in any publications.

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