Anti-Histone H2A.X antibody - ChIP Grade (ab47503)

Overview

  • Product nameAnti-Histone H2A.X antibody - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description
    Rabbit polyclonal to Histone H2A.X - ChIP Grade
  • SpecificityThis antibody detects endogenous levels of total Histone H2A.X protein. ab47503 reacts with non-phosphorylated form of Histone H2A.X and not the phosphorylated form of the protein.
  • Tested applicationsChIP, ICC/IF, ELISA, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse
  • Immunogen

    A sinthetic non-phosphopeptide derived from human Histone H2A.X around the phosphorylation site of serine 140 (Q-A-SP-Q-E)

  • Positive control
    • Hela cell extracts; Human breast carcinoma tissue. IHC-P: Human normal colon FFPE tissue sections. ICC/IF: HeLa cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab47503 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
ChIP Use 2 µg for 25 µg of chromatin.
ICC/IF Use a concentration of 1 µg/ml.
ELISA 1/10000.
WB 1/500 - 1/1000. Detects a band of approximately 42 kDa (predicted molecular weight: 15 kDa).
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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
    • 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
    • RGD1566119 antibody
    see all

Anti-Histone H2A.X antibody - ChIP Grade images

  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab47503 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • IHC image of ab47503 staining Histone H2A.X in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab47503, 1µg/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. No primary antibody was used in the negative control (shown on the inset).

     

    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.

  • ab47503 staining Histone H2A.X in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab47503 at 1μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green). AlexaFluor®350 WGA was used at a 1/200 dilution and incubated for 1h with the cells, to label plasma membranes (shown in blue). Nuclear DNA was labelled in red with 1.25 μM DRAQ5™ (ab108410), which was added to the secondary antibody mixture. A secondary only negative control is displayed, which indicates that the Histone H2A.X staining observed is due to primary antibody specificity and not to unspecific binding of the secondary antibody to the cells.

  • Lanes 1 - 2 : Anti-Histone H2A.X antibody - ChIP Grade (ab47503) at 1/500 dilution
    Lanes 3 - 4 : Anti-Histone H2A.X (phospho S140) antibody (ab47371) at 1/500 dilution

    Lane 1 : Extracts from Hela cells treated with UV
    Lane 2 : Extracts from Hela cells treated with UV with blocking peptide
    Lane 3 : Extracts from Hela cells
    Lane 4 : Extracts from Hela cells treated with UV


    Predicted band size : 15 kDa
    Observed band size : 15 kDa
  • Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using ab47503 at a 1/50 dilution.

    Left image : Untreated

    Right image : Treated with PeptideImmunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using ab47503 at a 1/50 dilution.

    Left image : Untreated

    Right image : Treated with Peptide

References for Anti-Histone H2A.X antibody - ChIP Grade (ab47503)

ab47503 has not yet been referenced specifically in any publications.

Product Wall

There are currently no Abreviews or Questions for ab47503.
Please use the links above to contact us or submit feedback about this product.

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