Anti-gamma H2A.X (phospho S139) antibody (ab11174)

Overview

  • Product nameAnti-gamma H2A.X (phospho S139) antibodySee all gamma H2A.X primary antibodies ...
  • Description
    Rabbit polyclonal to gamma H2A.X (phospho S139)
  • SpecificityUsing IF, this antibody was shown to bind to a non-nuclear location in Hela cells.
  • Tested applicationsIHC-P, ICC/IF, WB, Flow Cyt, ICCmore details
  • Species reactivity
    Reacts with: Mouse, Chicken, Human
    Predicted to work with: Rabbit, Guinea pig, Cow, Dog, Pig, Rhesus monkey, Gorilla, Chinese Hamster, Bat
  • Immunogen

    Synthetic phospho-peptide, which represents a portion of the C-terminus of human histone H2AX surrounding phosphorylated serine 139 (LocusLink ID 3014).

  • Positive control
    • Tested with human HEK293, human G-361 and mouse embryonic fibroblast cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab11174 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
IHC-P 1/1000 - 1/5000.
ICC/IF Use at an assay dependent concentration.
WB 1/2000 - 1/10000. Detects a band of approximately 15 kDa.
Flow Cyt Use 0.5µg for 106 cells.
ICC 1/1000 - 1/5000.
  • Application notesIs unsuitable for IP.
  • 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 S139) antibody images

    • Immunocytochemistry/Immunofluorescence analysis of neocarzinostatin treated asynchronous HeLa cells (left) and untreated asynchronous HeLa cells (right) labelling H2A.X (phospho S139 with ab11174 at 1/5000 (0.2µg/ml). A DyLight® 594-conjugated anti-rabbit IgG (1/100) was used as the secondary antibody.
    • All lanes : Anti-gamma H2A.X (phospho S139) antibody (ab11174) at 1/5000 dilution

      Lane 1 : HeLa nuclear lysate - untreated
      Lane 2 : HeLa nuclear lysate - IR treated

      Lysates/proteins at 40 µg per lane.

      Secondary
      HRP-conjugated donkey anti-rabbit IgG polyclonal
      developed using the ECL technique

      Performed under reducing conditions.

      Observed band size : 17 kDa (why is the actual band size different from the predicted?)


      Exposure time : 30 seconds

      This image is courtesy of an anonymous Abreview

      See Abreview

    • Samples: Nuclear extract (50 µg) from human HEK293, human melanoma (G361), mouse wildtype embryonic fibroblasts (+/+) or mouse H2AX knockout embryonic fibroblasts (-/-). Antibody: ab11174 used at 0.1 mcg/ml. Detection: Chemiluminescence with 30 second exposure.

      UT = untreated
      Ub = ubiquitylated
      NCS = neocarzinostatin - 200 ng/ml, 30 min

       

      Samples: Nuclear extract (50 µg) from human HEK293, human melanoma (G361), mouse wildtype embryonic fibroblasts (+/+) or mouse H2AX knockout embryonic fibroblasts (-/-). Antibody: ab11174 used at 0.1 mcg/ml. Detection: Chemiluminescence with 30 second exposure.

      UT = untreated
      Ub = ubiquitylated
      NCS = neocarzinostatin - 200 ng/ml, 30 min

    • Detection of gamma-H2AX by Immunofluorescence. Samples: Wildtype (H2AX +/+) or H2AX knockout (H2AX -/-) mouse embryonic fibroblasts. Antibody: Affinity purified ab11174 used at 2ug/ml. Detection: Rhodamine red labelled goat anti-rabbit IgG.Detection of gamma-H2AX by Immunofluorescence. Samples: Wildtype (H2AX +/+) or H2AX knockout (H2AX -/-) mouse embryonic fibroblasts. Antibody: Affinity purified ab11174 used at 2ug/ml. Detection: Rhodamine red labelled goat anti-rabbit IgG.
    • ab11174 at 1/1000 staining human HeLa cells by ICC/IF. These cells express a gene that causes a DNA damage response, leading to H2AX phosphorylation. The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 45 minutes. An Alexa-Fluor ®  488 conjugated goat anti-rabbit was used as the secondary.

      See Abreview

    • ab11174 at 0.5ug staining gamma H2A.X in human Jurkat cell line by flow cytometery. Cells were treated with 5ug/ml etopside for 3 hours, fixed in 1.5% paraformaldehyde, and permeabilized in 90% methanol. A FITC-conjugated rabbit polyclonal was used as secondary (in 150ul reaction). The black line indicates cells treated with etopside and anti KLH antibody, the red line repersents untreated cells and anti gamma H2A.X and blue line indicates etopside treated anti gamma H2A.X respectively.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling gamma H2A.X (phospho S139) with ab11174 at 1/5000 (0.2µg/ml). Dectection: DAB.

    References for Anti-gamma H2A.X (phospho S139) antibody (ab11174)

    This product has been referenced in:
    • Vasudevarao MD  et al. Naphthoquinone-mediated inhibition of lysine acetyltransferase KAT3B/p300, basis for non-toxic inhibitor synthesis. J Biol Chem 289:7702-17 (2014). WB . Read more (PubMed: 24469461) »
    • Sutherland JM  et al. Developmental expression of Musashi-1 and Musashi-2 RNA-binding proteins during spermatogenesis: analysis of the deleterious effects of dysregulated expression. Biol Reprod 90:92 (2014). IHC-P ; Mouse . Read more (PubMed: 24671879) »

    See all 24 Publications for this product

    Product Wall

    Application Western blot
    Loading amount 40 µg
    Gel Running Conditions Reduced Denaturing (4-12%)
    Sample Human Cell lysate - nuclear (HeLa)
    Specification HeLa
    Treatment IR
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
    Username

    Abcam user community

    Verified customer

    Submitted Apr 22 2014

    Application Western blot
    Loading amount 20 µg
    Gel Running Conditions Reduced Denaturing (15%)
    Sample Human Cell lysate - whole cell (U2OS osteosarcoma)
    Specification U2OS osteosarcoma
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Username

    Abcam user community

    Verified customer

    Submitted Feb 28 2014

    Application Immunocytochemistry/ Immunofluorescence
    Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
    Sample Human Cell (T98G Human brain glioblastoma)
    Specification T98G Human brain glioblastoma
    Permeabilization Yes - 0.1% v/v Triton X-100 pH 7.4 for 5 min at RT
    Fixative Paraformaldehyde
    Username

    Dr. Dimitra Kalamida

    Verified customer

    Submitted Dec 12 2013

    Application Western blot
    Loading amount 20 µg
    Gel Running Conditions Reduced Denaturing (15%)
    Sample Mouse Cell lysate - nuclear (smooth muscle cells)
    Specification smooth muscle cells
    Treatment 40uM t-Bhp for 1 hour followed by recovery
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Username

    Abcam user community

    Verified customer

    Submitted Jul 10 2013

    Application Immunocytochemistry
    Sample Human Cultured Cells (glioma)
    Specification glioma
    Fixative Paraformaldehyde
    Permeabilization Yes - 0,3 % triton x
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    Submitted Mar 21 2013

    Application Western blot
    Sample Human Cell lysate - whole cell (brain)
    Loading amount 60 µg
    Specification brain
    Treatment Irradiation 0, 2, 4,6 Gy for 24 h
    Gel Running Conditions Reduced Denaturing (4-12 % gel)
    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 Mar 18 2013

    ro complete our questionnaire.

    The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality

    I appreciate the time you have spent in ...

    Read More

    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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    Thank you for your message and for your patience in waiting for a response. I can confirm that ab11175 will only bind H2AX that is not phosphorylated on the Ser 139 i.e. it does not appear to bind to gamma H2AX. Blast search shows that it shoul...

    Read More
    Application Immunocytochemistry/ Immunofluorescence
    Sample Human Cell (HeLa)
    Specification HeLa
    Fixative Formaldehyde
    Permeabilization Yes - Methanol, 30 min at -20°C
    Blocking step FCS as blocking agent for 30 minute(s) · Concentration: 20% · Temperature: RT°C
    Username

    Dr. Ioannis Gavvovidis

    Verified customer

    Submitted Dec 02 2010

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