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    hif-1-alpha-antibody-h1alpha67-ab1.pdf

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Cardiovascular Hypoxia HIF
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Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

  • Datasheet
  • SDS
Reviews (44)Q&A (13)References (335)

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Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
  • Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
  • Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
  • Flow Cytometry - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

Key features and details

  • Mouse monoclonal [H1alpha67] to HIF-1 alpha
  • Suitable for: IP, Flow Cyt, ICC/IF, WB
  • Reacts with: Human
  • Isotype: IgG2b

Conjugates logo Related conjugates and formulations

Alexa Fluor® 647

You may also be interested in

Protein
Product image
Recombinant Human HIF-1 alpha protein (ab154478)
Primary
Product image
Alexa Fluor® 647 Anti-HIF-1 alpha antibody [H1alpha67] (ab203848)
Knockout
Product image
Human HIF1A (HIF-1 alpha) knockout HCT116 cell line (ab255394)

View more associated products

Overview

  • Product name

    Anti-HIF-1 alpha antibody [H1alpha67]
    See all HIF-1 alpha primary antibodies
  • Description

    Mouse monoclonal [H1alpha67] to HIF-1 alpha
  • Host species

    Mouse
  • Tested applications

    Suitable for: IP, Flow Cyt, ICC/IF, WBmore details
    Unsuitable for: IHC-Fr or IHC-P
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HeLa (DFO treated 0.5mM, 24h) nuclear lysate (ab180880); Human whole cell lysate (human lung adenocarcinoma cell line ADLC-5M2) (DFO treated 100uM, 16h). IP: HeLa (DFO treated 0.5 mg) nuclear lysate. ICC: MCF7 cells. Flow Cyt: HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 1mM Deferoxamine (ab120727) for 24 hours.
  • General notes

    For WB, we recommend using positive control samples such as DFO or CoCl2 treated nulcear cell lysates such as ab180880. Ensure cell lysis occurs quickly (within 2 mins) if removed from hypoxia. Loading a high amount of sample (>50 µg) and addition of protease inhibitors (e.g. ab65621) may also enhance detection.

    This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: PBS, 6.97% L-Arginine
  • Concentration information loading...
  • Purity

    Protein G purified
  • Clonality

    Monoclonal
  • Clone number

    H1alpha67
  • Isotype

    IgG2b
  • Research areas

    • Cardiovascular
    • Hypoxia
    • HIF
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • HLH / Leucine Zipper
    • HLH
    • Epigenetics and Nuclear Signaling
    • Cardiovascular/Immune
    • Hypoxia
    • HIF
    • Cancer
    • Invasion/microenvironment
    • Hypoxia
    • HIF
    • Cancer
    • Cancer Metabolism
    • Response to hypoxia
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Nucleotide metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia
    • Metabolism
    • Types of disease
    • Cancer
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Alexa Fluor® 647 Anti-HIF-1 alpha antibody [H1alpha67] (ab203848)
  • Assay kits

    • HIF-1 alpha Transcription Factor Assay Kit (ab133104)
  • ChIP Related Products

    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
  • Isotype control

    • Mouse IgG2b, kappa monoclonal [7E10G10] - Isotype Control (ab170192)
  • Positive Controls

    • Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880)
  • Recombinant Protein

    • Recombinant Human HIF-1 alpha protein (ab154478)
    • Recombinant Human HIF-1 alpha protein (ab48734)
  • Related Products

    • Prestained Protein Ladder – Broad molecular weight (10-245 kDa) (ab116028)
    • Metformin hydrochloride, AMPK activator (ab120847)
    • HeLa nuclear extract lysate (ab150036)
    • Human HIF-1 alpha ELISA Kit (ab171577)
    • HIF-1 alpha (H1alpha67, EP1215Y, ESEE122, EPR16897) Antibody Panel (ab263460)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab1 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
IP (2)
Use a concentration of 5 µg/ml.
Flow Cyt
Use at an assay dependent concentration.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

ICC/IF (6)
1/100 - 1/200.

PubMed: 25422886

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody.

WB (22)
Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).

We recommend blocking for 1 hour with 5% milk in TBST and reducing to 2% milk in TBST for the primary and secondary antibody incubation steps. For primary antibody incubation, we recommend 2 hours at room temperature.

We recommend Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody.

 

Notes
IP
Use a concentration of 5 µg/ml.
Flow Cyt
Use at an assay dependent concentration.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

ICC/IF
1/100 - 1/200.

PubMed: 25422886

We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody.

WB
Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).

We recommend blocking for 1 hour with 5% milk in TBST and reducing to 2% milk in TBST for the primary and secondary antibody incubation steps. For primary antibody incubation, we recommend 2 hours at room temperature.

We recommend Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody.

 

Application notes
Is unsuitable for IHC-Fr or IHC-P.

Target

  • 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.
  • Target information above from: UniProt accession Q16665 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3091 Human
    • Entrez Gene: 15251 Mouse
    • Entrez Gene: 29560 Rat
    • Omim: 603348 Human
    • SwissProt: Q16665 Human
    • SwissProt: Q61221 Mouse
    • SwissProt: O35800 Rat
    • Unigene: 597216 Human
    • Unigene: 3879 Mouse
    • Unigene: 446610 Mouse
    • Unigene: 10852 Rat
    see all
  • Alternative names

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

Images

  • Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    All lanes : Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 5 µg/ml

    Lane 1 : HeLa nuclear extract lysate (ab150036) at 40 µg
    Lane 2 : Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880) at 40 µg
    Lane 3 : HeLa nuclear control at 40 µg
    Lane 4 : HeLa nuclear DFO treated at 40 µg
    Lane 5 : Recombinant Human HIF-1 alpha protein (ab154478) at 0.001 µg

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 92 kDa


    Exposure time: 20 minutes


    We recommend using 5% milk in TBST as the blocking agent, decreasing to 2% milk in TBST during primary and secondary antibody incubation.

    Blots were developed with Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody

     

  • Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Immunoprecipitation - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

    HIF-1 alpha was immunoprecipitated using 0.5 mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5 µg of Mouse monoclonal to HIF-1 alpha and 50 µl of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.

    Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70°C; 10 µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1.

    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1:20,000 dilution.

    Band: 110 kDa; HIF1 alpha

  • Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)This image is taken from an Abreview submitted by Mike Campa, no further information is known about this image
    Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 1/400 dilution + Human whole cell lysate (human lung adenocarcinoma cell line ADLC-5M2) treated for 16 hours with 100 micromolar deferoxamine (DFO) at 20 µg

    Performed under reducing conditions.

    Predicted band size: 92 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?



    PVDF membrane was used and blocked for 16 hours in 5% milk.

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

    ab1 staining HIF-1-alpha in MCF7 (Human breast adenocarcinoma cell line) cells treated with metformin hydrochloride ab12084, by ICC/IF. Decrease in HIF-1-alpha expression correlates with increased concentration of metformin hydrochloride, as described in literature.
    The cells were incubated at 37°C for 24 hours in media containing different concentrations of ab120847 (metformin hydrochloride) in water, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2 hours at room temperature. Staining of the treated cells with ab1 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)This Image is courtesy of an anonymous Abreview

    ab1 at 1/200 dilution staining HIF-1 alpha in human 293FT cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde, permeabilized in 0.5% Trition X-100 and blocked in 5% BSA for 1 hour at 25°C. The primary antibody was used at 1/200 dilution in PBS and incubated with sample at 4°C for 12 hours. An Alexa Fluor® 488 conjugated Goat polyclonal to mouse IgG was used as secondary at 1/500 dilution.

    See Abreview

  • Flow Cytometry - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
    Flow Cytometry - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)

    Flow cytometry using ab1. HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were cultured untreated or with 1mM Deferoxamine (ab120727) for 24 hours to induce HIF-1-alpha protein levels. Cells were then trypsinized, fixed with paraformaldehyde and stained with ab1 (0.5 µg/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-Fluor® 488 dye. Unstained (black), untreated (red) and DFO treated (blue) cell traces are shown.

Protocols

  • Nuclear Extract Preparation for HIF 1 alpha
  • Western blot protocol advice for ab1

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (335)

Publishing research using ab1? Please let us know so that we can cite the reference in this datasheet.

ab1 has been referenced in 335 publications.

  • Wen M  et al. HIF-1a mediates the protective effect of plasma extracellular particles induced by remote ischaemic preconditioning on oxidative stress injury in human umbilical vein endothelial cells. Exp Ther Med 23:48 (2022). PubMed: 34917179
  • Liu Y  et al. Effects of blood oxygen saturation on pulmonary artery remodeling in an in vitro perfusion circuit model. J Thorac Dis 13:2169-2176 (2021). PubMed: 34012567
  • Liu H  et al. MicroRNA-210-3p is transcriptionally upregulated by hypoxia induction and thus promoting EMT and chemoresistance in glioma cells. PLoS One 16:e0253522 (2021). PubMed: 34197482
  • He M  et al. ALDH2/SIRT1 Contributes to Type 1 and Type 2 Diabetes-Induced Retinopathy through Depressing Oxidative Stress. Oxid Med Cell Longev 2021:1641717 (2021). PubMed: 34725563
  • Zheng B  et al. Lidocaine Alleviates Sepsis-Induced Acute Lung Injury in Mice by Suppressing Tissue Factor and Matrix Metalloproteinase-2/9. Oxid Med Cell Longev 2021:3827501 (2021). PubMed: 34804364
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-2 of 2 Abreviews or Q&A

Immunoprecipitation abreview for Anti-HIF-1 alpha antibody [H1alpha67] - ChIP Grade

Good
Abreviews
Abreviews
abreview image
Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (Hela)
Total protein in input
60 µg
Treatment
1% Hypoxia
Immuno-precipitation step
Protein G
Specification
Hela
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Aug 14 2017

Immunoprecipitation abreview for Anti-HIF-1 alpha antibody [H1alpha67] - ChIP Grade

Good
Abreviews
Abreviews
abreview image
Application
Immunoprecipitation
Sample
Mouse Tissue lysate - whole (Mouse brain hippocampal tissues)
Total protein in input
300 µg
Immuno-precipitation step
Protein G
Specification
Mouse brain hippocampal tissues
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Apr 07 2017

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