MitoSciences (MS1099)

H2A.X (pSer139) Human In-Cell ELISA Kit (IR) (ab131382)

Overview

  • Product nameH2A.X (pSer139) Human In-Cell ELISA Kit (IR)
  • Detection methodIR
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    HeLa Cells < 10%
    HeLa Cells < 10%
  • Tests
    1 x 96 well plate
  • Sample type
    Adherent cells, Suspension cells
  • Assay typeCell-based (qualitative)
  • Assay durationMultiple steps standard assay
  • Species reactivity
    Reacts with: Human
  • Product overview

    The H2A.X phospho Ser139 Human In-Cell ELISA Kit (IR) (ab131382) is designed to study the induction of DNA damage in response to various stimuli. A rabbit monoclonal antibody specific to H2A.X phospho Ser139 is used in this high-throughput duplexing plate-based assay. H2A.X phospho Ser139 is a reliable readout for double-stranded DNA breaks.

     

    Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.

  • Tested applicationsIn-Cell ELISAmore details
  • PlatformMicroplate

Properties

  • 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-H2AX 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 (By similarity). 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. Ubiquitination at Lys-14 and Lys-16 (H2AK13Ub and H2AK15Ub, respectively) in response to DNA damage is initiated by RNF168 that mediates monoubiquitination at these 2 sites, and 'Lys-63'-linked ubiquitin are then conjugated to monoubiquitin; RNF8 is able to extend 'Lys-63'-linked ubiquitin chains in vitro. H2AK119Ub and ionizing radiation-induced 'Lys-63'-linked ubiquitination (H2AK13Ub and H2AK15Ub) are distinct events.
    Acetylation at Lys-37 increases in S and G2 phases. This modification has been proposed to play a role in DNA double-strand break repair.
  • Cellular localizationNucleus. Chromosome.
  • Information by UniProt
  • Alternative names
    • H2A histone family, member X
    • H2A.X
    • H2a/x
    • H2AFX
    • H2AX
    • H2AX histone
    • H2AX_HUMAN
    • Histone H2A.X
    • Histone H2AX
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab131382 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
In-Cell ELISA Use at an assay dependent concentration.

H2A.X (pSer139) Human In-Cell ELISA Kit (IR) images

  • Sample experiment using ab131382 on HeLa cells following drug treatment: H2A.X (pSer139) readout. HeLa cells were treated for 4 hours with dose titrations of Camptothecin, Etoposide and Staurosporin. The dashed grey line indicates the vehicle control signal.
  • Specificity of H2A.X (pSer139) antibodies demonstrated by immunocytochemistry. The primary antibody used in this assay kit was validated by staining HeLa cells treated with 10 µM Camptothecin or vehicle for 4 hours and imaged by fluorescent microscopy. Note the absence of H2A.X (pSer139) in the untreated cells.
  • All lanes : H2A.X (pSer139) Human In-Cell ELISA Kit (IR) (ab131382)

    Lane 1 : Untreated cells
    Lane 2 : Camptothecin treated cells
    Lane 3 : UV exposed cells
  • Antibody specificity demonstrated by Western Blot Analysis: H2A.X (pSer139) is phospho-specific. Jurkat cells were stimulated with UV light exposure to induce H2A.X phospho Ser139 and then the UV treated lysate was treated with lambda protein phosphatase. Top panel: H2A.X (pSer139) is induced by UV treatment and the western blot band is sensitive to phosphatase treatment. Lower panel: In contrast, total H2A.X (ab124781) levels are not sensitive to phosphatase treatment.

Protocols

References for H2A.X (pSer139) Human In-Cell ELISA Kit (IR) (ab131382)

ab131382 has not yet been referenced specifically in any publications.

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