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

製品の概要

  • 製品名
    Anti-gamma H2A.X (phospho S139) antibody
    gamma H2A.X 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to gamma H2A.X (phospho S139)
  • 特異性
    Using IF, this antibody was shown to bind to a non-nuclear location in Hela cells.
  • アプリケーション
    適用あり: IHC-P, ICC/IF, WB, Flow Cyt, ICCmore details
    適用なし: IP
  • 種交差性
    交差種: Mouse, Chicken, Human, Dictyostelium discoideum
    交差が予測される動物種: Rabbit, Guinea pig, Cow, Dog, Pig, Rhesus monkey, Gorilla, Chinese hamster, Bat
  • 免疫原

    Synthetic peptide corresponding to Human gamma H2A.X (phospho S139).
    Database link: 3014
    (Peptide available as ab40211)

  • ポジティブ・コントロール
    • Tested with human HEK293, human G-361 and mouse embryonic fibroblast cells.

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
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.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

ICC 1/1000 - 1/5000.
  • 追加情報
    Is unsuitable for IP.
  • ターゲット情報

    • 機能
      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.
    • 配列類似性
      Belongs to the histone H2A family.
    • 発生段階
      Synthesized in G1 as well as in S-phase.
    • ドメイン
      The [ST]-Q motif constitutes a recognition sequence for kinases from the PI3/PI4-kinase family.
    • 翻訳後修飾
      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.
    • 細胞内局在
      Nucleus. Chromosome.
    • Information by UniProt
    • 参照データベース
    • 別名
      • H2A histone family member X antibody
      • 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

    画像

    • 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

    • 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.
    • 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.

    • 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.

    • 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

    • 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

    参考文献

    This product has been referenced in:
    • Maierhofer A  et al. Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation. PLoS One 12:e0177442 (2017). ICC/IF ; Human . Read more (PubMed: 28489894) »
    • Kotzur T  et al. Granulocyte colony-stimulating factor (G-CSF) promotes spermatogenic regeneration from surviving spermatogonia after high-dose alkylating chemotherapy. Reprod Biol Endocrinol 15:7 (2017). ICC/IF ; Mouse . Read more (PubMed: 28077131) »

    See all 71 Publications for this product

    レビューと Q&A

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Ovarian Cancer cell line OVCA420)
    Permeabilization
    Yes - 0.3% Triton
    Specification
    Ovarian Cancer cell line OVCA420
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
    Fixative
    Methanol
    Username

    Ms. Hsiao-Wang Chen

    Verified customer

    投稿 Oct 21 2017

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Western blot
    Sample
    Cow Cell lysate - whole cell (vascular smooth muscle cells)
    Gel Running Conditions
    Reduced Denaturing (10% gel)
    Loading amount
    30 µg
    Treatment
    1uM etoposide
    Specification
    vascular smooth muscle cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    投稿 Jul 21 2017

    Application
    Western blot
    Loading amount
    10000 cells
    Gel Running Conditions
    Reduced Denaturing (4-12% gradient)
    Sample
    Human Cell lysate - whole cell (huh7 liver)
    Specification
    huh7 liver
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    投稿 Dec 30 2014

    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 - Triton X-100
    Fixative
    Formaldehyde
    Username

    Abcam user community

    Verified customer

    投稿 Sep 02 2014

    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

    投稿 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

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    投稿 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

    投稿 Dec 12 2013

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

    投稿 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

    投稿 Mar 21 2013

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

    投稿 Mar 18 2013

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