Overview

  • Product name
    Anti-HIF1 beta antibody [2B10]
    See all HIF1 beta primary antibodies
  • Description
    Mouse monoclonal [2B10] to HIF1 beta
  • Host species
    Mouse
  • Specificity
    Detects aryl hydrocarbon (Ah) receptor nuclear translocator (ARNT).
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, ICC, IHC-P, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Xenopus laevis, Fish, Non human primates, Zebrafish, African green monkey
    Predicted to work with: Rabbit, Cow
  • Immunogen

    Synthetic peptide corresponding to Human HIF1 beta aa 771-789.
    Sequence:

    NSYNNEEFPDLTMFPPFSE

Properties

Applications

Our Abpromise guarantee covers the use of ab2771 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
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

EMSA Use at an assay dependent concentration.
ICC/IF 1/1000.
ICC Use at an assay dependent concentration.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IP Use at an assay dependent concentration.
WB 1/4000. Detects a band of approximately 87 kDa (predicted molecular weight: 86 kDa).

Target

  • Function
    Required for activity of the Ah (dioxin) receptor. This protein is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after ligand binding. The complex then initiates transcription of genes involved in the activation of PAH procarcinogens. The heterodimer with HIF1A or EPAS1/HIF2A functions as a transcriptional regulator of the adaptive response to hypoxia.
  • 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.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • arnT antibody
    • ARNT protein antibody
    • ARNT_HUMAN antibody
    • Aryl hydrocarbon receptor nuclear translocator antibody
    • bHLHe2 antibody
    • Class E basic helix-loop-helix protein 2 antibody
    • Dioxin receptor antibody
    • Dioxin receptor nuclear translocator antibody
    • Drnt antibody
    • HIF 1 beta antibody
    • HIF 1beta antibody
    • HIF-1-beta antibody
    • HIF1-beta antibody
    • HIF1B antibody
    • HIF1beta antibody
    • Hypoxia Inducible Factor 1 antibody
    • Hypoxia inducible factor 1 beta antibody
    • Hypoxia-inducible factor 1-beta antibody
    • nuclear translocator antibody
    • Tango antibody
    see all

Images

  • Immunocytochemistry/Immunofluorescence analysis of HIF1 beta shows staining in A2058 cells. HIF1 beta (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2771 (1:100) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of HIF1 beta shows staining in HeLa cells. HIF1 beta (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2771 (1:20) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of HIF1 beta shows staining in U251 cells. HIF1 beta (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2771 (1:100) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Anti-HIF1 beta antibody [2B10] (ab2771) at 1/2000 dilution + Human U2OS cells - whole cell lysate at 20 µg

    Secondary
    HRP conjugated goat anti-mouse antibody

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 86 kDa
    Observed band size: 90 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 50 kDa (possible non-specific binding)


    Exposure time: 5 minutes

    See Abreview

  • IHC image of ab2771 staining in human tonsil formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2771, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Overlay histogram showing PC12 cells stained with ab2771 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2771, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in PC12 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Shih JW  et al. Long noncoding RNA LncHIFCAR/MIR31HG is a HIF-1a co-activator driving oral cancer progression. Nat Commun 8:15874 (2017). WB ; Human . Read more (PubMed: 28639619) »
  • Guo F  et al. Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells. Cell Res 27:967-988 (2017). ICC/IF ; Mouse . Read more (PubMed: 28621329) »
See all 11 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Answer

While we do have publications using this antibody in mouse samples we are not presently aware of specific publications using mouse brain homogenates. HIF1 beta forms heterodimers with HIF1alpha and has ubiquitous nuclear expression. The HIF1 complex rapidly degrades. It is critical to prep only a few plates/dishes/flasks of cells at a time and to immediately get the cells into ice cold buffers and perform the whole protein prep on ice. HIF-1 is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% or with treat using certain agents (CoCl2, DFO, etc.) so proper sample preparation is critical. Upon stabilization HIF-1 translocates to the nucleus. The best western blots (cleanest) are always done using nuclear extracts. It is possible to detect HIF-1 in whole cell extracts, but they tend to be much dirtier and the staining is much weaker. We recommend that a positive/negative control always be run side by side so that it is possible to discern which band is upregulated in the hypoxic sample. Depending on the sample, treatment, etc. you may see either a band or a doublet.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HOS cells)
Loading amount
50 µg
Specification
HOS cells
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Mar 03 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (293T)
Total protein in input
500 µg
Specification
293T
Immuno-precipitation step
Protein G

Abcam user community

Verified customer

Submitted Oct 30 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
African Green Monkey Cell lysate - whole cell (Cos-7 cell line)
Loading amount
30 µg
Specification
Cos-7 cell line
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Sep 26 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (3T3 cell line)
Loading amount
30 µg
Specification
3T3 cell line
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Sep 26 2007

Question

BATCH NUMBER 271685 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Double bands, MW is around 90.For human and rat samples, the bands from homogenate are Darker than from extract opposite for mouse samples. The bands from mouse samples are much darker than the others. SAMPLE I tested the antibody with brain samples such as human homogenate, human extract, rat homogenate, rat extract, mouse homogenate and mouse extract. PRIMARY ANTIBODY HIF1 beta ab2771, 271685. 1:1000 diluted, in 5% non-fat milk incubate for 18 hours,overnight, room temperature wash with 0.1% tween-TBS for 3 times, at 10 minutes each time DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED not used ANTIBODY STORAGE CONDITIONS -20, but +4 degrees before use diluted (1:4) in 5%BSA. SAMPLE PREPARATION in SDS buffer containing 2%SDS, 10%glycerol, 1%beta-mercaptoethanol and 0.0625M Tris-Cl AMOUNT OF PROTEIN LOADED 10ug ELECTROPHORESIS/GEL CONDITIONS 10% reducing TRANSFER AND BLOCKING CONDITIONS buffer:1.4% glycine, 0.3% Trizma base, 20% methanol 1 hour block with 5% non-fat milk SECONDARY ANTIBODY goat anti-mouse 1:5000 diluted in 5% non-fat milk for 2 hours at room temperature wash with 0.1% tween-TBS for 3 times, at 10 minutes each time HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? not altered ADDITIONAL NOTES I checked literature and found HIF1 beta has several isoforms. Does ab2771 recognize only one isoforms or all? I failed to attach the image of the film, which is jpg or doc format.

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Answer

Thank you for your patience. I am sorry to hear that you are experiencing difficulties with this product ab2771 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. In most cases, the cause of multiple bands is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. You can try decreasing the primary (1:1000 ~ 1:5000) and secondary concentration or run a no-primary control (without the primary antibody) to ensure that the multiple bands are not caused by your secondary antibody. Another possible reason is that the extra bands you are observing may be multimers. Please try boiling your protein in SDS-PAGE for 10 minutes to disrupt multimers. Meanwhile, bands located below the expected molecular weight you are observing may indicate that your target protein has been digested. Please make sure that you incorporate sufficient protease inhibitors in your sample buffer. The image you provided showed different strengths in expression. I would suggest doing optimization for each species sample separately, so that you can have better control of the level and intensity of the bands. This is because your target protein may exist in different amount in different species. I would also suggest incubating your primary antibody at 4 degrees instead of at room temperature. Incubation at room temperature is recommended to be done for 1-2 hours, while 4 degrees can be done overnight. Unfortunately, I do not have any data suggesting that this product can detect other HIF-1 beta isoforms at this time. However, I believe the following reference may help explain the double band that you are observing. Rescue of Hypoxia-inducible Factor-1 alpha-deficient Tumor Growth by Wild-Type Cells Is Independent of Vascular Endothelial Growth Factor. Can. Res. 62(10):2962-2970, 2002. http://cancerres.aacrjournals.org/cgi/reprint/62/10/2962 In this specific reference, the authors detect nonspecific cross-reactivity of the anti-ARNT polyclonal antibody in nuclear extracts of ES cells. Also, we see similar reactivity in the reference: Isoform specific expression of hypoxia-inducible factor-1alpha during the late stages of mouse spermiogenesis. Mol. Endocrinol., Vol 16: 234, Feb 2002. http://mend.endojournals.org/cgi/reprint/16/2/234 I hope the above suggestions and references will be helpful to you. If you still experience problems please do not hesitate to contact me with details of your order (purchase order number, shipping address/purchasing agent info, contact information, etc.) so that I can immediately arrange for a replacement or refund to you. Also, please advice on how you would to proceed with this enquiry. I look forward to hearing from you again in order to resolve the matter.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (forebrain)
Loading amount
15 µg
Specification
forebrain
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 15 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jun 22 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Cell lysate - whole cell (PC12)
Loading amount
20 µg
Specification
PC12
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 21 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (U2OS)
Loading amount
20 µg
Specification
U2OS
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 15 2007

Answer

Thank you for your enquiry. Followingf corresponce with the source of this antibody I have determined that the cell lines used for IP were mouse Hep1c1c7 and human HeLa cells. ab2771 works well by IP in both mouse and human lysates at 1-2 ug/IP. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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1-10 of 12 Abreviews or Q&A

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