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HIF-1 alpha Transcription Factor Assay Kit (ab133104)

Submit a review Q&A (2)References (17)
Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit
  • Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit
  • Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit
  • Functional Studies - HIF-1 alpha Transcription Factor Assay Kit (ab133104)

Key features and details

  • Assay type: Semi-quantitative
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Sample type: Cell culture extracts, Cell Lysate

Overview

  • Product name

    HIF-1 alpha Transcription Factor Assay Kit
    See all HIF-1 alpha kits

  • Detection method

    Colorimetric

  • Sample type

    Cell culture extracts, Cell Lysate

  • Assay type

    Semi-quantitative

  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Predicted to work with: Mammals

  • Product overview

    HIF-1 alpha Transcription Factor Assay (ab133104) is a non-radioactive, sensitive method for detecting specific transcription factor DNA binding activity in nuclear extracts and whole cell lysate.


    A 96-well enzyme-linked immunosorbent assay (ELISA) replaces the cumbersome radioactive electrophoretic mobility shift assay (EMSA). A specific double stranded DNA (dsDNA) sequence containing the HIF-1 alpha response element (5’-ACGTG-3’) is immobilized to the wells of a 96-well plate. HIF-1 alpha contained in a nuclear extract, binds specifically to the HIF-1 alpha response element. The HIF transcription factor complex is detected by addition of a specific primary antibody directed against HIF-1 alpha. A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 96 tests
    96-Well Plate Cover 1 unit
    Polysorbate 20 1 vial
    Transcription Factor Antibody Binding Buffer (10X) 1 x 3ml
    Transcription Factor Binding Assay Buffer (4X) 1 x 3ml
    Transcription Factor Developing Solution 1 x 12ml
    Transcription Factor Goat Anti-Rabbit HRP Conjugate 1 x 100µl
    Transcription Factor HIF-1 alpha 96-Well Strip Plate 1 unit
    Transcription Factor HIF-1 alpha Competitor dsDNA 1 vial
    Transcription Factor HIF-1 alpha Positive Control 1 vial
    Transcription Factor HIF-1 alpha Primary Antibody 1 vial
    Transcription Factor Reagent A 1 x 120µl
    Transcription Factor Stop Solution 1 x 12ml
    Wash Buffer Concentrate (400X) 1 x 5ml

  • 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

    see all

Images

  • Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit
    Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit

    HEK293 cells were treated with 1 mM deferoxamine mesylate (DFO) for 1 or 24 hours. 40 micoliters of nuclear lysates (ab113474; corresponding to 4e6 cells) were tested in duplicates (+/- SD).

  • Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit
    Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit

    Titration of positive control with different volumes of inhibitor (TA), background signal subtracted (duplicates; +/- SD).

  • Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit
    Functional Assay: ab133104 HIF-1 alpha Transcription Factor Assay Kit

    Titration of positive control, background signal subtracted (duplicates; +/- SD).

  • Functional Studies - HIF-1 alpha Transcription Factor Assay Kit (ab133104)
    Functional Studies - HIF-1 alpha Transcription Factor Assay Kit (ab133104)
    Assay of nuclear extract from stimulated HeLa cells (100 µM CoCl2) and non-stimulated HeLa cells.

References (17)

Publishing research using ab133104? Please let us know so that we can cite the reference in this datasheet.

ab133104 has been referenced in 17 publications.

  • Cenigaonandia-Campillo A  et al. Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer. Theranostics 11:3595-3606 (2021). PubMed: 33664850
  • Dong F  et al. Hypoxia-dependent expression of MAP17 coordinates the Warburg effect to tumor growth in hepatocellular carcinoma. J Exp Clin Cancer Res 40:121 (2021). PubMed: 33832535
  • Bi Z  et al. Nrf2 and HIF1a converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells. Theranostics 10:4134-4149 (2020). PubMed: 32226544
  • Logan SM  et al. The brains of six African mole-rat species show divergent responses to hypoxia. J Exp Biol 223:N/A (2020). PubMed: 32041803
  • Yang W  et al. Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity. Nat Commun 11:4457 (2020). PubMed: 32901017
View all Publications for this product

Customer reviews and Q&As

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1-2 of 2 Abreviews or Q&A

Question

I only have 200,000 cells per sample, rather than the 10 million that you recommend. What is the lower detection limit of the assay? Do you think my samples will meet this limit with 200,000 cells?

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Answer

We typically recommend to start with one million cells, but satisfactory results of this assay are influenced by the cell type and treatment to stimulate HIF-1α. To give you a baseline idea of how much sample protein is necessary in our hands, the detection limit of this assay is approximately 5 µg of 100 µM CoCl2 stimulated HeLa nuclear extract.

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Question


I would like to use the HIF-1 alpha transcription factor assay kit (ab133104) but before I need to perform a nuclear extraction. I was looking in your website and found this product ab113474. Can I do a nuclear extraction using ab113474 and then do my experiment with ab133104?

Read More
Answer



Yes you can use ab113474, EpiSeeker Nuclear Extraction Kit, to purify soluble nuclear proteins from tissue culture cells or tissues and then detect HIF1 alpha in that extract using ab133104, HIF-1 alpha Transcription Factor Assay Kit.

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