Anti-Histone H2A.X antibody - ChIP Grade (ab11175)

Overview

  • Product name
    Anti-Histone H2A.X antibody - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description
    Rabbit polyclonal to Histone H2A.X - ChIP Grade
  • Tested applications
    Suitable for: IHC-P, WB, IP, ChIP, ICC/IF, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Monkey
    Predicted to work with: Rabbit, Rhesus monkey, Gorilla
  • Immunogen

    This information is considered to be commercially sensitive.

  • Positive control
    • Tested with human HEK293, human G-361 and mouse embryonic fibroblast cells.
  • General notes
    The phosphorylated form of this Ab is known as gamma H2A.X, when phosphorylated at Ser 139.

Properties

Applications

Our Abpromise guarantee covers the use of ab11175 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 Use at an assay dependent concentration.
WB 1/5000 - 1/15000. Detects a band of approximately 15 kDa.
IP Use at 5-20 µg/mg of lysate.
ChIP Use 2 µg for 25 µg of chromatin. PubMed: 19380460
ICC/IF 1/2500. (see review). Use periodate-lysine-PFA fixative.
IHC-Fr 1/1000.

Target

  • Function
    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.
  • Sequence similarities
    Belongs to the histone H2A family.
  • Developmental stage
    Synthesized in G1 as well as in S-phase.
  • Domain
    The [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 localization
    Nucleus. 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
    • Histone H2AX antibody
    • RGD1566119 antibody
    see all

Images

  • Samples: Nuclear extract from human 293T, human G-361, and wild-type (+/+) or H2AX knockout (-/-) mouse embryonic fibroblasts. Antibody: ab11175 used at 0.1 mcg/ml. Detection: Chemiluminescence with an exposure time of 5 seconds.

    Samples: Nuclear extract from human 293T, human G-361, and wild-type (+/+) or H2AX knockout (-/-) mouse embryonic fibroblasts. Antibody: ab11175 used at 0.1 mcg/ml. Detection: Chemiluminescence with an exposure time of 5 seconds.

  • ICC/IF image of ab11175 stained Hela 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 (ab11175, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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.

  • ab11175 staining Histone H2A.X in mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% BSA for 60 minutes at 25°C; antigen retrieval was by heat mediation in a Na-citrate pH6 buffer. Samples were incubated with primary antibody (1/100 in PBS/Triton) for 3 hours at 25°C. A Alexa Flour® 555-conjugated donkey anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody.

    See Abreview

  • 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 ab11175 (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.

  • ab11175 staining Histone H2A.X in Mouse testis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 4% BSA for 2 hours at 37°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in diluent) for 48 hours at 4°C. ab6564 Goat polyclonal anti-Rabbit IgG - H&L Cy5® (1/300) was used as the secondary antibody.

    Staining left to right:
    1) DAPI;
    2) Histone H2A.X;
    3) Merge

    See Abreview

References

This product has been referenced in:
  • Xiong H  et al. Twist1 Enhances Hypoxia Induced Radioresistance in Cervical Cancer Cells by Promoting Nuclear EGFR Localization. J Cancer 8:345-353 (2017). WB ; Human . Read more (PubMed: 28261334) »
  • Su WP  et al. Chronic treatment with cisplatin induces chemoresistance through the TIP60-mediated Fanconi anemia and homologous recombination repair pathways. Sci Rep 7:3879 (2017). WB ; Human . Read more (PubMed: 28634400) »

See all 78 Publications for this product

Customer reviews and Q&As

Thank you for contacting us.

ab11175 and ab18255 were never used togteher in Sandwich ELISA. These however can be used in Indirect ELISA.

The sELISA application totally based on difference in epiotopes, the antibodies recognize mea...

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The lots GR4642-4 and GR4642-6 stem from the same original batch ("mother lot"), and the only difference is that they have been aliquoted on a different day. But all other characteristics are the same. <

In our experience this product appears to have a preference for the H2AX lacking phosphorylation at S139. However, it does appear to bind gamma H2AX to some degree.

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...

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The constituents of the Tris/Citrate/Phosphate buffer are: 150mM Tris 60mM Citrate 8mM Phosphate

The immunogen for this antibody is proprietary information. However, the peptide starts at 121 aa and goes toward the phosphorylated site.

This has not been rigorously tested. Preliminary indications are that Ab11175 has considerably higher affinity for H2AX than it does for phosphorylated H2AX.

Gel:16% PAGE, Laemli system MW markers: RPN-800 (Amersham)

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

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