RabMAb

Anti-Histone H2A.X (phospho S139) antibody [EP854(2)Y] - ChIP Grade (ab81299)

Overview

  • Product nameAnti-Histone H2A.X (phospho S139) antibody [EP854(2)Y] - ChIP GradeSee all Histone H2A.X primary antibodies ...
  • Description
    Rabbit monoclonal [EP854(2)Y] to Histone H2A.X (phospho S139) - ChIP Grade
  • Tested applicationsWB, IHC-P, ICC/IF, CHIPseq, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    A synthetic phospho-peptide corresponding to residues surrounding serine 139 of Human Histone H2A.X protein was used as the immunogen.

  • Positive control
    • Jurkat cell lysate and Human kidney transitional cell carcinoma.
  • General notes

    Produced under U.S. Patent No. 5,675,063.

    This product is available conjugated to DyLight® 488 see ab126708.

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab81299 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 1/100000 - 1/500000. Predicted molecular weight: 15 kDa.
IHC-P 1/100 - 1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/50 - 1/100.
CHIPseq Use at an assay dependent concentration. PubMed: 20714222
Flow Cyt 1/100.

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 (phospho S139) antibody [EP854(2)Y] - ChIP Grade images

  • Overlay histogram showing HeLa cells stained with ab81299 (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 (ab81299, 1/100 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) (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.
  • IHC-P image of γH2A.X staining on Mouse testis sections using ab81299 (1:5000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab81299 was diluted 1:5000 in TBS buffer (containing BSA and Azide) and sections were then incubated with ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

    See Abreview

  • IHC-P image of γH2A.X staining on Rat testis sections using ab81299 (1:5000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab81299 was diluted 1:5000 in TBS buffer (containing BSA and Azide) and sections were then incubated with ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

    See Abreview

  • All lanes : Anti-Histone H2A.X (phospho S139) antibody [EP854(2)Y] - ChIP Grade (ab81299) at 1/500000 dilution

    Lane 1 : Jurkat cell lysates Jurkat cell lysates
    Lane 2 : Jurkat cell lysates treated with etoposide

    Lysates/proteins at 10 µg per lane.

    Secondary
    HRP Labelled goat anti-rabbit at 1/2000 dilution

    Predicted band size : 15 kDa
    Observed band size : 15 kDa
  • ab81299, at 1/100 staining Human kidney transitional cell carcinoma by Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney transitional cell carcinoma using 1/100 ab81299.
  • ICC/IF image of ab81299 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab81299, neat) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References for Anti-Histone H2A.X (phospho S139) antibody [EP854(2)Y] - ChIP Grade (ab81299)

This product has been referenced in:
  • Xie C  et al. Expression of ?H2AX in various gastric pathologies and its association with Helicobacter pylori infection. Oncol Lett 7:159-163 (2014). IHC ; Human . Read more (PubMed: 24348841) »
  • Ha GH  et al. TACC3 deregulates the DNA damage response and confers sensitivity to radiation and PARP inhibition. Oncogene 0:N/A (2014). Human . Read more (PubMed: 24769898) »

See all 14 Publications for this product

Product Wall

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 15 minute(s) · Concentration: 0.1% · Temperature: 37°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate
Sample Mouse Tissue sections (skin)
Specification skin
Permeabilization No
Fixative formalin
Username

Abcam user community

Verified customer

Submitted Jun 03 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Non-reduced Denaturing (12% gel)
Sample Human Cell lysate - whole cell (HEK293)
Specification HEK293
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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Abcam user community

Verified customer

Submitted Apr 25 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Sample Human Cell (HEK293)
Specification HEK293
Permeabilization Yes - triton
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 24 2014

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

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Testis)
Specification Testis
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization No
Blocking step BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Username

Mr. Carl Hobbs

Verified customer

Submitted May 15 2013

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (Testis)
Specification Testis
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization No
Blocking step BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Username

Mr. Carl Hobbs

Verified customer

Submitted May 15 2013

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Rat Tissue sections (Testis)
Specification Testis
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization No
Blocking step BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Username

Mr. Carl Hobbs

Verified customer

Submitted May 15 2013

Thank you for your enquiry.

Could you confirm this is product number ab81299?

I have investigated our records for this antibody ab81299 with my colleagues and can confirm that regrettably there has been an error. This antibody has...

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