Anti-gamma H2A.X (phospho S140) antibody [3F2] (ab22551)

Overview

  • Product nameAnti-gamma H2A.X (phospho S140) antibody [3F2]
    See all gamma H2A.X primary antibodies
  • Description
    Mouse monoclonal [3F2] to gamma H2A.X (phospho S140)
  • Tested applicationsWB, ICC/IF, IHC-P, ELISA, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Other Immunogen Type corresponding to Human gamma H2A.X. Synthetic peptide sequence surrounding phosphorylated Ser140

  • Positive control
    • Gamma irradiated HeLA cell lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab22551 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 Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa.
ICC/IF Use a concentration of 2 - 4 µg/ml.
IHC-P Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.

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
    • 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 S140) antibody [3F2] images

  • ab22551 labelling gamma H2A.X (phospho S140) in HeLa cells by immunocytochemistry/immunofluorescence.

  • ab22551 labelling gamma H2A.X (phospho S140) in control (left) and G2/M Arrested (right) cells by Immunocytochemistry/Immunofluorescence.

  • Western blot analysis of Phospho-H2A.X pSer140(ab22551) at a concentration of 1 ug/ml on Jurkat cell untreated (Lane 1) and Jurkat cell stimulated with staurosporine (Lane 2) followed by HRP conjugated goat anti-mouse lgG (H+L) Secondary Antibody.

  • All lanes : Anti-gamma H2A.X (phospho S140) antibody [3F2] (ab22551)

    Lane 1 : gamma irradiated HeLa cells: Unphosphorylated H2AX control
    Lane 2 : gamma irradiated HeLa cells: Phosphorylated H2AX
  • Immunofluorescence detection of phosphorylated H2AX in gamma irradiated HeLa cells using ab22551. Immunofluorescence detection of phosphorylated H2AX in gamma irradiated HeLa cells using ab22551.
  • IHC image of ab22551 staining in Human breast formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab22551, 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.

    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.

  • ICC/IF image of ab22551 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 (ab22551, 10µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) 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.

  • Overlay histogram showing HeLA cells stained with ab22551 (red line). The cells were fixed with 100% 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 (ab22551, 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 Mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • A431 cell nucleus (DAPI signal) showing three gamma-H2AX foci structures.
    Primary anitibody H2A.X (phospho S140) antibody [3F2], ab22551, 100ug.
    Secondary antibody Mouse IgG-Fc (FITC), ab97264, 1mg.

References for Anti-gamma H2A.X (phospho S140) antibody [3F2] (ab22551)

This product has been referenced in:
  • Slatter TL  et al. Smoking during pregnancy causes double-strand DNA break damage to the placenta. Hum Pathol 45:17-26 (2014). IHC-P ; Human . Read more (PubMed: 24125744) »
  • Guo P  et al. MiR-26a enhances the radiosensitivity of glioblastoma multiforme cells through targeting of ataxia-telangiectasia mutated. Exp Cell Res 320:200-8 (2014). WB . Read more (PubMed: 24211747) »

See all 25 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (HeLa cell lysate)
Loading amount 40 µg
Specification HeLa cell lysate
Treatment +/- 10 Gy IR
Gel Running Conditions Reduced Denaturing (4-12% Bis-Tris)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted May 07 2013

Application Western blot
Loading amount 15 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (vascular smooth muscle cell)
Specification vascular smooth muscle cell
Treatment 1 uM doxorubicin for 3 hours
Blocking step Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted Nov 25 2014

Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Sample Human Cell (HeLa)
Specification HeLa
Permeabilization Yes - 0.1% Triton X-100
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jun 04 2013

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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Thank you for your reply and for kindly confirming these details.

The details you have kindly provided will provide us with vital information for our monitoring of product quality

I appreciate the time you have spent in the labora...

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Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this...

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Thank you for contacting us. There will be a slight immunogen difference between each antibody. However, each recognizes the same portion of the targe,t gamma H2A.X (phospho S139), and should work in your experiments. Two of these antibodies are ra...

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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 (Skin tumor)
Specification Skin tumor
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer pH 6.0
Permeabilization Yes - TBST
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Username

Dr. Teo Manestar-Blazic

Verified customer

Submitted Sep 15 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Testis)
Specification Testis
Fixative Formaldehyde
Permeabilization Yes - 0.1% triton X-100
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1.5% · Temperature: 37°C
Username

Mr. Frédéric Leduc

Verified customer

Submitted Jan 22 2009

Application Immunocytochemistry/ Immunofluorescence
Sample Rat Cell (Testis)
Specification Testis
Fixative Formaldehyde
Permeabilization Yes - 0.1% triton X-100
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1.5% · Temperature: 37°C
Username

Mr. Frédéric Leduc

Verified customer

Submitted Jan 22 2009

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