Overview

  • Product name

    Anti-HIRA/HIR antibody [EPR7416]
    See all HIRA/HIR primary antibodies
  • Description

    Rabbit monoclonal [EPR7416] to HIRA/HIR
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IP, Flow Cyt, ICC/IFmore details
    Unsuitable for: IHC-P
  • Species reactivity

    Reacts with: Human
  • Immunogen

    within Human HIRA/HIR aa 950 to the C-terminus. The exact sequence is proprietary.
    Database link: P54198

  • Positive control

    • 293T, HeLa and K562 cell lysates, HeLa cells
  • General notes

     This product was previously labelled as HIRA

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab129169 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
WB 1/1000 - 1/10000. Predicted molecular weight: 112 kDa.

We recommend to use 1%SDS Hot lysis prepare method. For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

IP 1/10 - 1/100.
Flow Cyt 1/100 - 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

ICC/IF 1/50 - 1/100.
  • Application notes
    Is unsuitable for IHC-P.
  • Target

    • Function

      Cooperates with ASF1A to promote replication-independent chromatin assembly. Required for the periodic repression of histone gene transcription during the cell cycle. Required for the formation of senescence-associated heterochromatin foci (SAHF) and efficient senescence-associated cell cycle exit.
    • Tissue specificity

      Expressed at high levels in kidney, pancreas and skeletal muscle and at lower levels in brain, heart, liver, lung, and placenta.
    • Sequence similarities

      Belongs to the WD repeat HIR1 family.
      Contains 8 WD repeats.
    • Developmental stage

      Expressed during embryogenesis.
    • Post-translational
      modifications

      Sumoylated.
      Phosphorylated by CDK2/CCNA1 and CDK2/CCNE1 on Thr-555 in vitro. Also phosphorylated on Thr-555 and Ser-687 in vivo.
    • Cellular localization

      Nucleus. Nucleus > PML body. Primarily, though not exclusively, localized to the nucleus. Localizes to PML bodies immediately prior to onset of senescence.
    • Information by UniProt
    • Database links

    • Alternative names

      • DGCR1 antibody
      • DiGeorge critical region gene 1 antibody
      • HIR antibody
      • HIR histone cell cycle regulation defective homolog A antibody
      • HIRA antibody
      • HIRA protein antibody
      • HIRA_HUMAN antibody
      • Histone cell cycle regulator antibody
      • Protein HIRA antibody
      • TUP 1 antibody
      • TUP1 like enhancer of split protein 1 antibody
      • TUP1-like enhancer of split protein 1 antibody
      • TUPLE1 antibody
      see all

    Images

    • All lanes : Anti-HIRA/HIR antibody [EPR7416] (ab129169) at 1/1000 dilution

      Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
      Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
      Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

      Predicted band size: 112 kDa
      Observed band size: 112 kDa


      Exposure time: 3 minutes


      We recommend to use 1%SDS Hot lysis prepare method.

      We are unsure how to define the extra bands.

    • Overlay histogram showing HEK293 cells stained with ab129169 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab129169, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HEK293 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
    • ab129169, at 1/50 dilution, staining HIRA/HIR in HeLa cells by Immunofluorescence.

    • Equilibrium disassociation constant (KD)
      Learn more about KD

      Click here to learn more about KD

    References

    This product has been referenced in:

    • Zhu Z  et al. PHB Associates with the HIRA Complex to Control an Epigenetic-Metabolic Circuit in Human ESCs. Cell Stem Cell 20:274-289.e7 (2017). WB ; Human . Read more (PubMed: 27939217) »
    See 1 Publication for this product

    Customer reviews and Q&As

    1-10 of 15 Abreviews or Q&A

    Application
    Immunoprecipitation
    Sample
    Human Cell lysate - nuclear (MRC5)
    Total protein in input
    100000 cells
    Immuno-precipitation step
    Protein A/G
    Specification
    MRC5

    Abcam user community

    Verified customer

    Submitted Apr 03 2019

    Application
    ChIP
    Sample
    Human Cell lysate - nuclear (MRC5)
    Negative control
    IgG control
    Specification
    MRC5
    Detection step
    Real-time PCR
    Type
    Cross-linking (X-ChIP)
    Duration of cross-linking step: 15 minute(s) and 0 second(s)
    Specification of the cross-linking agent: PFA
    Positive control
    GAPDH

    Abcam user community

    Verified customer

    Submitted Apr 02 2019

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HT1080)
    Gel Running Conditions
    Non-reduced Denaturing (12)
    Loading amount
    100000 cells
    Specification
    HT1080
    Blocking step
    BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C

    Abcam user community

    Verified customer

    Submitted Mar 25 2019

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (MRC5)
    Permeabilization
    Yes - Triton X100
    Specification
    MRC5
    Blocking step
    BSA as blocking agent for 30 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 18°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Mar 25 2019

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    Yes - 0.5% Triton X-100
    Specification
    HeLa
    Fixative
    Paraformaldehyde

    Dr. Kirk Mcmanus

    Verified customer

    Submitted Aug 10 2018

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HAP1)
    Permeabilization
    Yes - 0.5% Triton 5 minutes
    Specification
    HAP1
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 24°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Feb 13 2018

    Answer

    Thank you for that confirmation. I wanted to be sure you were confident the NP40 was sufficient, since other antibodies will give the same result if it is not.

    I can send a replacement but we do not have an alternative lot of ab129169 and I am concerned that ab20655, our other HIRA antibody that has been tested in this application, lacks specificity, judging from the western blot results:

    Click here (or use the following: https://www.abcam.com/index.html?datasheet=20655).

    If you prefer a refund, please just let me know. It will be a credit applied to the card that was used to make the purchase.

    Read More

    Answer

    Thank you for the extra details of your protocol. You should be seeing a nuclear signal from this cell line, if the permeabilization is sufficient. Have you been able to stain other nuclear proteins with this protocol? We recommend fixation for no more than 15 minutes in 4% PFA, and permeabilization with 0.5% Triton X-100. I am wondering if your NP40 solution is not permeabilizing the nuclear membrane, since low concentrations can be used to isolate intact nuclei from whole cells.

    Read More

    Answer

    A replacement or refund for anti-HIRA ab129169 is available, assuming the species of your samples is human and not mouse or rat, which we list on the datasheet as non-reactive.

    For complaints, we also routinely ask for a few details of the protocol, as there may be something we can suggest to improve it for this antibody. Can you please describe how the samples are prepared, and the steps of the staining , for example, how are the samples blocked before applying the antibody, and what was the dilution or concentration of the antibody? Are you confident your other reagents are effective?

    I look forward to your reply.

    Read More

    Answer

    Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

    I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 11111111111.

    To check the status of the order please contact our Customer Service team and reference this number.

    Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

    I wish you the best of luck with your research.

    Read More

    1-10 of 15 Abreviews or Q&A

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