Overview

  • Product nameAnti-Histone H2A.X antibody - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description
    Rabbit polyclonal to Histone H2A.X - ChIP Grade
  • Tested applicationsChIP, WB, IP, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within amino acid residues 100 to the C-terminus of Human H2A.X.

  • Positive control
    • This antibody gave a positive signal in the following lysates: Mouse Lung, Mouse Testis, Rat Lung, Rat Testis, Colcemid treated recombinant histone H2A.X, Untreated recombinant histone H2A.X This antibody gave a positive result in IHC in the following FFPE tissue: Human Lung and human colon.

Properties

Applications

Our Abpromise guarantee covers the use of ab20669 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
ChIP Use 2 µg for 25 µg of chromatin.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human gamma H2A.X (unmodified ) peptide (ab15646).
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
IHC-P Use a concentration of 0.2 - 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • FunctionVariant 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 similaritiesBelongs to the histone H2A family.
  • Developmental stageSynthesized in G1 as well as in S-phase.
  • DomainThe [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 localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
  • 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
    • RGD1566119 antibody
    see all

Anti-Histone H2A.X antibody - ChIP Grade images

  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab20669 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • IHC image of ab20669 staining Histone H2A.X in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab20669, 0.5µg/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. No primary antibody was used in the negative control (shown on the inset).

     

    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.

  • ab20669 staining Histone H2A.X in Mouse brain neurone cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 5% serum for 2 hours at 22°C. Samples were incubated with primary antibody (1/500 in 2.5% serum + 0.3% Triton X-100 in PBS) for 24 hours at 4°C. ab175651, an Alexa Fluor® 405-conjugated Donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • IHC image of Histone H2A.X staining in Human Lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab20669, 0.2 µg/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.

  • All lanes : Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg
    Lane 2 : HeLa Histone Preparation Nuclear Lysate at 2.5 µg
    Lane 3 : Histone H2A Recombinant Protein (negative control) at 0.1 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)


    Exposure time : 20 minutes
  • ab20669 (1/2000) staining Histone H2A.X in Hela cells (green). Cells were fixed in methanol and counter-stained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.

    See Abreview

  • Histone H2A.X was immunoprecipitated using 0.5mg Rat Testis whole tissue extract, 5µg of Rabbit polyclonal to Histone H2A.X and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Rat Testis whole tissue extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab20669.
    Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
    Band: 17kDa: Histone H2A.X.
  • All lanes : Anti-Histone H2A.X antibody - ChIP Grade (ab20669) at 1 µg/ml

    Lane 1 : Lung (Mouse) Tissue Lysate
    Lane 2 : Testis (Mouse) Tissue Lysate
    Lane 3 : Lung (Rat) Tissue Lysate
    Lane 4 : Testis (Rat) Tissue Lysate - normal tissue (ab29388)

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 55 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 8 minutes

References for Anti-Histone H2A.X antibody - ChIP Grade (ab20669)

This product has been referenced in:
  • Lee JH  et al. Histone deacetylase inhibitor induces DNA damage, which normal but not transformed cells can repair. Proc Natl Acad Sci U S A 107:14639-44 (2010). WB, ICC/IF ; Human . Read more (PubMed: 20679231) »

See 1 Publication for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Sample Mouse Cell (Brain, neurons)
Specification Brain, neurons
Permeabilization Yes - 0.3% Triton
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 19 2013

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (4-12% gradient gel)
Sample Human Cell lysate - other (lymphocytes)
Specification lymphocytes
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 37°C
Username

Abcam user community

Verified customer

Submitted Nov 21 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (293T cell)
Loading amount 30 µg
Specification 293T cell
Gel Running Conditions Reduced Denaturing (4-12% Gradient Gel)
Blocking step Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Nov 10 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Methanol
Permeabilization No
Username

Dr. Kirk McManus

Verified customer

Submitted Oct 13 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (10T1/2)
Specification 10T1/2
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% Triton X100 in PBS
Username

Dr. Kirk McManus

Verified customer

Submitted Sep 12 2008

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"