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  1. Link

    histone-h2ax-antibody-nuclear-marker-ab10475.pdf

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Epigenetics and Nuclear Signaling Histones Variants
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Validated using a knockout cell line

Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)

  • Datasheet
  • SDS
Submit a review Q&A (1)References (8)

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Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
  • Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
  • Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)

Key features and details

  • Rabbit polyclonal to Histone H2A.X - Nuclear Marker
  • Suitable for: WB, IHC-P
  • Knockout validated
  • Reacts with: Cow, Human
  • Isotype: IgG

Get better batch-to-batch reproducibility with a recombinant antibody

Product image
Anti-Histone H2A.X antibody [EPR895] (ab124781)
  • Research with confidence – consistent and reproducible results with every batch
  • Long-term and scalable supply – powered by recombinant technology for fast production
  • Success from the first experiment – confirmed specificity through extensive validation
  • Ethical standards compliant – production is animal-free

Overview

  • Product name

    Anti-Histone H2A.X antibody - Nuclear Marker
    See all Histone H2A.X primary antibodies
  • Description

    Rabbit polyclonal to Histone H2A.X - Nuclear Marker
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Cow, Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • General notes

    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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Histones
    • Variants
    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • DNA Damage Response
    • DNA Damage Recognition
    • Epigenetics and Nuclear Signaling
    • Histones
    • H2A
    • Unmodified

Associated products

  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    •  Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • Recombinant Protein

    • Recombinant Human Histone H2A.X protein (ab134863)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab10475 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
1/500 - 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Notes
WB
1/500 - 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function

    Variant histone H2A which replaces conventional H2A in a subset of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation and for efficient repair of DNA double strand breaks (DSBs) specifically when modified by C-terminal phosphorylation.
  • Sequence similarities

    Belongs to the histone H2A family.
  • Developmental stage

    Synthesized in G1 as well as in S-phase.
  • Domain

    The [ST]-Q motif constitutes a recognition sequence for kinases from the PI3/PI4-kinase family.
  • Post-translational
    modifications

    Phosphorylated on Ser-140 (to form gamma-H2AFX or H2AX139ph) in response to DNA double strand breaks (DSBs) generated by exogenous genotoxic agents and by stalled replication forks, and may also occur during meiotic recombination events and immunoglobulin class switching in lymphocytes. Phosphorylation can extend up to several thousand nucleosomes from the actual site of the DSB and may mark the surrounding chromatin for recruitment of proteins required for DNA damage signaling and repair. Widespread phosphorylation may also serve to amplify the damage signal or aid repair of persistent lesions. Phosphorylation of Ser-140 (H2AX139ph) in response to ionizing radiation is mediated by both ATM and PRKDC while defects in DNA replication induce Ser-140 phosphorylation (H2AX139ph) subsequent to activation of ATR and PRKDC. Dephosphorylation of Ser-140 by PP2A is required for DNA DSB repair. In meiosis, Ser-140 phosphorylation (H2AX139ph) may occur at synaptonemal complexes during leptotene as an ATM-dependent response to the formation of programmed DSBs by SPO11. Ser-140 phosphorylation (H2AX139ph) may subsequently occurs at unsynapsed regions of both autosomes and the XY bivalent during zygotene, downstream of ATR and BRCA1 activation. Ser-140 phosphorylation (H2AX139ph) may also be required for transcriptional repression of unsynapsed chromatin and meiotic sex chromosome inactivation (MSCI), whereby the X and Y chromosomes condense in pachytene to form the heterochromatic XY-body. During immunoglobulin class switch recombination in lymphocytes, Ser-140 phosphorylation (H2AX139ph) may occur at sites of DNA-recombination subsequent to activation of the activation-induced cytidine deaminase AICDA. Phosphorylation at Tyr-143 (H2AXY142ph) by BAZ1B/WSTF determines the relative recruitment of either DNA repair or pro-apoptotic factors. Phosphorylation at Tyr-143 (H2AXY142ph) favors the recruitment of APBB1/FE65 and pro-apoptosis factors such as MAPK8/JNK1, triggering apoptosis. In contrast, dephosphorylation of Tyr-143 by EYA proteins (EYA1, EYA2, EYA3 or EYA4) favors the recruitment of MDC1-containing DNA repair complexes to the tail of phosphorylated Ser-140 (H2AX139ph).
    Monoubiquitination of Lys-120 (H2AXK119ub) by RING1 and RNF2/RING2 complex gives a specific tag for epigenetic transcriptional repression. Following DNA double-strand breaks (DSBs), it is ubiquitinated through 'Lys-63' linkage of ubiquitin moieties by the E2 ligase UBE2N and the E3 ligases RNF8 and RNF168, leading to the recruitment of repair proteins to sites of DNA damage. Monoubiquitination and ionizing radiation-induced 'Lys-63'-linked ubiquitination are distinct events.
  • Cellular localization

    Nucleus. Chromosome.
  • Target information above from: UniProt accession P16104 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 531733 Cow
    • Entrez Gene: 3014 Human
    • Omim: 601772 Human
    • SwissProt: P16104 Human
    • Unigene: 477879 Human
    • Alternative names

      • AW228881 antibody
      • H2A histone family member X antibody
      • H2A.FX antibody
      • H2A.X antibody
      • H2a/x antibody
      • H2AFX antibody
      • H2AX antibody
      • H2AX histone antibody
      • H2AX_HUMAN antibody
      • Hist5.2ax antibody
      • Histone 2A antibody
      • Histone 2AX antibody
      • Histone H2A.X antibody
      • Histone H2AX antibody
      • RGD1566119 antibody
      see all

    Images

    • Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
      Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
      All lanes : Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/500 dilution

      Lane 1 : Wild-type HAP1 whole cell lysate
      Lane 2 : H2AFX (Histone H2A.X) knockout HAP1 whole cell lysate
      Lane 3 : HEK293 whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 15 kDa



      Lanes 1 - 4: Merged signal (red and green). Green - ab10475 observed at 17 kDa. Red - loading control, ab9484, observed at 37 kDa.

      ab10475 was shown to specifically react with Histone H2A.X in wild-type HAP1 cells as signal was lost in H2AFX (Histone H2A.X) knockout cells. Wild-type and H2AFX (Histone H2A.X) knockout samples were subjected to SDS-PAGE. Ab10475 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
      Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
      Lanes 1 & 3 : Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/500 dilution
      Lane 2 : Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/1000 dilution

      Lanes 1-2 : Calf thymus histone lysate
      Lane 3 : Calf thymus histone lysate with Human Histone H2A.X (unmodified ) peptide (ab15020) at 1 µg

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

      Performed under reducing conditions.

      Predicted band size: 15 kDa


      Exposure time: 30 seconds
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)

      IHC image of Histone H2A X staining in human B cell lymphoma FFPE section, performed on a 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 ab10475, 1µg/ml, for 8 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.

    • Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
      Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)

      HeLa cells were incubated at 37°C for 3h with vehicle control (0 µM) and different concentrations of camptothecin (ab120115). Increased expression of γH2A.X (phospho S139) in HeLa cells correlates with an increase in camptothecin concentration, as described in literature.

      Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab2893 at 1 µg/ml and ab10475 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

    Protocols

    • Western blot protocols
    • Immunohistochemistry protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (8)

    Publishing research using ab10475? Please let us know so that we can cite the reference in this datasheet.

    ab10475 has been referenced in 8 publications.

    • Mehibel M  et al. Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models. J Clin Invest 131:N/A (2021). PubMed: 34060485
    • Sen N  et al. EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis. Mol Carcinog 57:1342-1357 (2018). PubMed: 29873416
    • Xiao G  et al. B-Cell-Specific Diversion of Glucose Carbon Utilization Reveals a Unique Vulnerability in B Cell Malignancies. Cell 173:470-484.e18 (2018). PubMed: 29551267
    • Ji J  et al. Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay. PLoS One 12:e0171582 (2017). ELISA, ICC/IF ; Human . PubMed: 28158293
    • York D  et al. Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53. Vet Comp Oncol 15:1087-1100 (2017). PubMed: 27333821
    • Zeng Q  et al. Overexpression of miR-155 promotes the proliferation and invasion of oral squamous carcinoma cells by regulating BCL6/cyclin D2. Int J Mol Med 37:1274-80 (2016). WB . PubMed: 26986233
    • Kim MK  et al. Loss of compensatory pro-survival and anti-apoptotic modulator, IKKe, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21. Oncotarget 5:12788-802 (2014). WB . PubMed: 25474241
    • Goldowitz D  et al. Molecular pathways underpinning ethanol-induced neurodegeneration. Front Genet 5:203 (2014). WB ; Mouse . PubMed: 25076964

    Customer reviews and Q&As

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

    Question

    Your datasheet shows that this product has been tested for ELISA, but I can't find anything in the literature showing an ELISA for H2AX.

    Read More

    Abcam community

    Verified customer

    Asked on Nov 02 2004

    Answer

    Thank you for your enquiry. Ab10475 was tested in a peptide ELISA as part of the antibody characterization process. If you have any more questions, please contact us again.

    Read More

    Abcam Scientific Support

    Answered on Nov 04 2004

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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