Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)

Overview

  • Product nameAnti-gamma H2A.X (phospho S139) antibody [9F3]See all gamma H2A.X primary antibodies ...
  • Description
    Mouse monoclonal [9F3] to gamma H2A.X (phospho S139)
  • Tested applicationsFlow Cyt, WB, IP, ICC/IF, IHC-P, In Situ Hybridisation more details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Monkey
  • Immunogen

    Synthetic peptide of phosphorylated (Ser139) human Histone H2A.X.

  • Positive control
    • HeLa heat shocked cell lysate for Western blot.

Properties

Applications

Our Abpromise guarantee covers the use of ab26350 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
Flow Cyt Use 1µg for 106 cells.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 16 kDa.
IP Use a concentration of 12.5 µg/ml.
ICC/IF 1/500.
IHC-P Use a concentration of 1 µg/ml.
In Situ Hybridisation Use at an assay dependent dilution.

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.
  • 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
  • Alternative names
    • H2A histone family member X antibody
    • H2A.FX antibody
    • H2A.X antibody
    • H2A/X antibody
    • H2AFX antibody
    • H2AX antibody
    • H2AX_HUMAN antibody
    • Histone H2A.x antibody
    see all

Anti-gamma H2A.X (phospho S139) antibody [9F3] images

  • All lanes : Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350) at 1/1000 dilution

    Lane 1 : Untreated Zebrafish embryos.
    Lane 2 : Zebrafish embryos treated with 25 Gy of X-Ray.
    Lane 3 : Zebrafish embryos treated with 50 Gy of X-Ray

    Secondary
    Goat anti-mouse HRP at 1/10000 dilution

    This image is courtesy of an anonymous Abreview

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  • Overlay histogram showing HeLa cells stained with ab26350 (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 (ab26350, 1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was a mix of mouse IgG1 [ICIGG1], (ab91353, 1μg/1x106 cells), IgG2a [ICIGG2A], (ab91361, 1μg/1x106 cells), IgG2b [PLPV219], (ab91366, 1μg/1x106 cells), IgG3 [MG3-35], (ab18394, 1μg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • ab26350 staining gamma H2A.X in pig spermatocytes by Immunocytochemistry/ Immunofluorescence. Cells were fixed with formaldehyde, permeabilized with Triton x100 and blocking with 0.15% BSA was performed for 30 minutes at 250C was performed. Samples were incubated with primary antibody (1/100: in PBS + 0.15% BSA+0.1% Tween 20) for 12 hours at 25°C. An Alexa Fluor®488-conjugated goat monoclonal to mouse IgG was used at dilution at 1/100 as secondary antibody.

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  • ab26350 (1µg/ml) staining gamma H2A in human spleen, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining .
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • ab26350 (1/500) staining gamma H2A.X (phospho S139) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

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  • All lanes : Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)

    Lane 1 : Molecular weight marker
    Lane 2 : Cell lysates prepared from human Jurkat cells
    Lane 3 : Cell lysates prepared from human Jurkat cells treated with staurosporine
    Lane 4 : Cell lysates prepared from mouse NIH3T3 cells
    Lane 5 : Cell lysates prepared from CHO-K1 cells
    Lane 6 : Cell lysates prepared from Rat-2 cells

References for Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)

This product has been referenced in:
  • Kracikova M  et al. A threshold mechanism mediates p53 cell fate decision between growth arrest and apoptosis. Cell Death Differ 20:576-88 (2013). Read more (PubMed: 23306555) »
  • Triscott J  et al. Personalizing the treatment of pediatric medulloblastoma: Polo-like kinase 1 as a molecular target in high-risk children. Cancer Res 73:6734-44 (2013). Read more (PubMed: 24019381) »

See all 5 Publications for this product

Product Wall

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Buffer/Enzyme Used: 0.01M citric acid/citrate sodium
Sample Mouse Tissue sections (adult testes)
Specification adult testes
Permeabilization No
Fixative 4% PFA
Username

Dr. Qing Wen

Verified customer

Submitted Apr 04 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Non-reduced Non-Denaturing (Native) (precast 4-15%)
Sample Human Cell lysate - whole cell (in leammli)
Specification in leammli
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Username

Abcam user community

Verified customer

Submitted Apr 03 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate ph6, 20 min 100C
Sample Human Tissue sections (cerebellum)
Specification cerebellum
Permeabilization No
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jul 15 2013

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (MCF7)
Specification MCF7
Fixative Formaldehyde
Permeabilization Yes - triton 1%
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Apr 15 2013


I have conducted a sequence alignment between the immunogen of ab26350 and the zebrafish histone H2AX however, the alignment was very poor. There was only 5% homology therefore unfortunately this antibody is not suitable for use in zebrafish.
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Es freut mich zu hören, dass Sie unseren Antikörper ab26350 in Gefrierschnitten (IHC-Fr) testen möchten. Sie können den Gutscheincode zum Kauf eines weiteren Antikörpers einsetzen, sobald Sie uns ein Abreview ...

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Thank you for your patience.

For ab26350:
The lab let me know that theyhave not specifically isotyped this antibody so they do not knowfor certain which subisotype the antibody is.We only know it is mouse IgG.

The according is...

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Thank you for your reply.

We have 2 mouse monoclonals: ab26350 (IgG, I will inquire about the subisotype with the lab) and ab22551 (IgG1). Both have been tested in ICC/IF with human samples.

I will let you know what I find out abou...

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Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (U2OS)
Specification U2OS
Fixative Formaldehyde
Permeabilization Yes - NP40
Blocking step BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted May 24 2012

Thank you for your enquiry.

I can confirm that this antibody has not yet been tested forH2AX detection in yeast.

I contacted the originator who did a BLAST against human H2AX protein sequence but there was no yeast found on the li...

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