Recombinant
RabMAb

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

Overview

  • Product name
    Anti-Histone H2A.X (phospho S139) antibody [EP854(2)Y] - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description
    Rabbit monoclonal [EP854(2)Y] to Histone H2A.X (phospho S139) - ChIP Grade
  • Tested applications
    Suitable for: IP, WB, IHC-P, ICC/IF, 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 100 to the C-terminus (phospho S139). A synthetic phospho-peptide corresponding to residues surrounding serine 139 of Human Histone H2A.X protein was used as the immunogen
    Database link: P16104

  • Positive control
    • WB: HepG2 and Jurkat cell lysates. IHC-P: Human kidney transitional cell carcinoma, human brain, rat and mouse testis. ICC/IF: H2O2 treated HeLa cells. IP: HepG2 cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    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.

    This product is a recombinant rabbit monoclonal antibody.

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
IP 1/40.
WB 1/5000 - 1/10000. Predicted molecular weight: 15 kDa.
IHC-P 1/50 - 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/250.
ChIP Use at an assay dependent concentration. PubMed: 20360682

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.
  • 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

Images

  • All lanes : Anti-Histone H2A.X (phospho S139) antibody [EP854(2)Y] - ChIP Grade (ab81299) at 1/100000 dilution

    Lane 1 : HepG2 cell lysate – treated with etoposide
    Lane 2 : HepG2 cell lysate – untreated

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size : 15 kDa

    Blocking buffer - 5% NFDM/TBST

    Diluting buffer - 1% BSA

  • IHC-P image of γH2A.X staining on Rat testis sections using unpurified 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 unpurified ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

    See Abreview

  • Dot blot analysis of Histone H2A.X single phospho peptide pS139 (lane 1) and Histone H2A.X non-phospho peptide (lane 2) with ab81299 at 1/1000. Blocking and diluting buffer was 5% NFDM/TBST. The secondary antibody used was ab97051 Peroxidase conjugated Goat Anti-Rabbit IgG, (H+L) at 1/100,000.

  • ab81299 at 1/40 immunoprecipitating Histone H2A.X (phospho S139) in HepG2 (human hepatocellular carcinoma epithelial) whole cell lysate observed at 15 KDa (lanes 1 and 2).

    Lane 1 (input): HepG2 treated with etoposide and TSA whole cell lysate 10μg

    Lane 2 (+): ab81299 + HepG2 treated with etoposide and TSA whole cell lysate

    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab81299 in HepG2 treated with etoposide and TSA

    For western blotting, ab81299 (Purified) at 1/200 dilution and ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1000).

    Blocking buffer and concentration: 5% NFDM/TBST.
    Diluting buffer and concentration: 5% NFDM /TBST.

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

    Lane 1 : 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
  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells (untreated and treated with H2O2) labelling Histone H2A.X (phospho S139) with ab81299 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue). 

    Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

  • IHC-P image of γH2A.X staining on Mouse testis sections using unpurified 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 unpurified ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

    See Abreview

  • Immunohistochemical staining of paraffin embedded human brain with purified ab81299 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

  • Immunohistochemical staining of paraffin embedded human brain with unpurified ab81299 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

  • Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney transitional cell carcinoma using unpurified ab81299 at a dilution of 1/100.

  • Ab81299 staining H2A.X in Human Testis tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in Citric acid. Samples were incubated with primary antibody (1/50 in TBS) for 2 hours at 21°C. A biotin conjugated Anti-Rabbit IgG (goat polyclonal) was used as the secondary antibody at a 1/250 dilution.

    See Abreview

References

This product has been referenced in:
  • Biehs R  et al. DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination. Mol Cell 65:671-684.e5 (2017). Read more (PubMed: 28132842) »
  • Gu X  et al. AID-associated DNA repair pathways regulate malignant transformation in a murine model of BCL6-driven diffuse large B-cell lymphoma. Blood 127:102-12 (2016). Flow Cyt ; Mouse . Read more (PubMed: 26385350) »

See all 31 Publications for this product

Customer reviews and Q&As

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: Na-citrate pH6
Sample
Mouse Tissue sections (Liver, P5)
Specification
Liver, P5
Permeabilization
Yes - Triton 0,05%
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Oct 07 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

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (human u2os cells)
Negative control
sample without dna damage
Specification
human u2os cells
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 15 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Username

Abcam user community

Verified customer

Submitted Jul 08 2016

Application
Western blot
Sample
Mouse Recombinant protein (C-terminus of H2A.X with full range of phosphoryla)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (Dot blot)
Loading amount
0.384 µg
Specification
C-terminus of H2A.X with full range of phosphoryla
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Peter Ellis

Verified customer

Submitted Jun 02 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - Trypsin
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 23°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 26 2016

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
Username

Abcam user community

Verified customer

Submitted Apr 25 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

Abcam guarantees this product to work in the species/application used in this Abreview.
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

1-10 of 12 Abreviews or Q&A

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

Sign up