Anti-Histone H2A.X antibody [EPR895] - ChIP Grade (ab124781)

Overview

  • Product nameAnti-Histone H2A.X antibody [EPR895] - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description
    Rabbit monoclonal [EPR895] to Histone H2A.X - ChIP Grade
  • Specificityab126781 does not work in ICC/IF on PFA fixed cells but does work on methanol fixed cells.
  • Tested applicationsSuitable for: WB, IP, IHC-P, ICC/IF, Flow Cyt, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human Histone H2A.X aa 1-100.

  • Positive control
    • WB: Raji and HEK293 cell lysates, fetal kidney, human heart, mouse and rat kidney tissue lysates. IHC-P: Human kidney and clear cell carcinoma tissues. ICC/IF: HeLa cells. IP: HeLa cell lysate.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    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.

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

Properties

Applications

Our Abpromise guarantee covers the use of ab124781 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. Detects a band of approximately 14 kDa (predicted molecular weight: 15 kDa).
IP Use a concentration of 5 µg/ml.
IHC-P 1/500 - 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

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

ICC/IF 1/1000.

ab126781 does not work on PFA fixed cells but does work on methanol fixed cells.

Flow Cyt Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration. PubMed: 22275873

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
    • Histone H2AX antibody
    • RGD1566119 antibody
    see all

Anti-Histone H2A.X antibody [EPR895] - ChIP Grade images

  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H2A.X with purified ab124781 at 1/100 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

  • All lanes : Anti-Histone H2A.X antibody [EPR895] - ChIP Grade (ab124781) at 1/3000 dilution (purified)

    Lane 1 : Raji cell lysate
    Lane 2 : HEK293 cell lysate
    Lane 3 : Mouse kidney tissue lysate
    Lane 4 : Rat kidney tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size : 15 kDa
    Observed band size : 14,22 kDa (why is the actual band size different from the predicted?)

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • All lanes : Anti-Histone H2A.X antibody [EPR895] - ChIP Grade (ab124781) at 1/2000 dilution (unpurified)

    Lane 1 : Raji cell lysate
    Lane 2 : HEK293 cell lysate
    Lane 3 : Mouse kidney tissue lysate
    Lane 4 : Rat kidney tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size : 15 kDa
    Observed band size : 14 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 22 kDa. We are unsure as to the identity of these extra bands.

    ~22kDa band may be the monoubiquitinated form of histone H2A

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • All lanes : Anti-Histone H2A.X antibody [EPR895] - ChIP Grade (ab124781) at 1/1000 dilution (unpurified)

    Lane 1 : Raji lysate
    Lane 2 : Fetal kidney lysate
    Lane 3 : Human heart lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    HRP labelled Goat anti Rabbit IgG at 1/2000 dilution

    Predicted band size : 15 kDa
    Observed band size : 14 kDa (why is the actual band size different from the predicted?)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Histone H2A.X with unpurified ab124781 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Histone H2A.X with purified ab124781 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H2A.X with purified ab124781 at a dilution of 1/1000. Cells were fixed with either 4% PFA (top) or 100% methanol (bottom) and permeabilized with 0.1% TritonX-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.

    ab7291 mouse anti-Tubulin (1/1000) followed by ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were used to label tubulin. DAPI was used as the nulear counterstain.

  • Histone H2A.X was immunoprecipitated using 5ug of ab124781 from 200ul of HeLa whole cell extract lysate diluted to 0.5mg/ml in RIPA and 50ul of Protein G magnetic beads. No antibody was added to the control (-).

    The antibody was incubated under agitation with the Protein G beads for 10min, HeLa whole cell 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 70°C. 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab124781 at 1ug/ml. The Secondary antibody was Mouse monoclonal SB62a Anti-Rabbit IgG light chain (HRP) (ab99697) at 1/10,000 dilution.

    Band: 15kDa; Histone H2A.X

References for Anti-Histone H2A.X antibody [EPR895] - ChIP Grade (ab124781)

This product has been referenced in:
  • Ramaekers CH  et al. RNF8-independent Lys63 poly-ubiquitylation prevents genomic instability in response to replication-associated DNA damage. PLoS One 9:e89997 (2014). WB ; Human . Read more (PubMed: 24587176) »
  • Tsunoda S  et al. Differential responses of SOD1-deficient mouse embryonic fibroblasts to oxygen concentrations. Arch Biochem Biophys 537:5-11 (2013). Mouse . Read more (PubMed: 23811199) »

See all 3 Publications for this product

Product Wall

Application Western blot
Sample Chicken Tissue lysate - whole (thymus homogenates)
Gel Running Conditions Reduced Denaturing (10-20% Tris/Glycine gel)
Loading amount 10 µg
Specification thymus homogenates
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Jenny Shoots

Verified customer

Submitted Jan 13 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Mouse Cell lysate - whole cell (embryonic stem cell)
Specification embryonic stem cell
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jul 01 2013

Thank you for contacting us and your interest in our products.

I have had a look through the antibodies we have directed against the H2A.X. Unfortunately, most are not likely to react with the target you are hoping to detect in Arabidopsis. T...

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