Anti-Histone H2A (phospho S129) antibody - ChIP Grade (ab15083)

Overview

  • Product nameAnti-Histone H2A (phospho S129) antibody - ChIP Grade
    See all Histone H2A primary antibodies
  • Description
    Rabbit polyclonal to Histone H2A (phospho S129) - ChIP Grade
  • Tested applicationsSuitable for: WB, ChIP, PepArrmore details
  • Species reactivity
    Reacts with: Saccharomyces cerevisiae, Schizosaccharomyces pombe
    Does not react with: Human
  • Immunogen

    Synthetic peptide corresponding to Saccharomyces cerevisiae Histone H2A aa 100 to the C-terminus (phospho S129) conjugated to Bovine Serum Albumin (BSA).
    Database link: P04911

  • Positive control
    • MMS treated S. cerevisiae (0.1% for 30 minutes).

Properties

Applications

Our Abpromise guarantee covers the use of ab15083 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
WB 1/500 - 1/1000. Detects a band of approximately 14 kDa (predicted molecular weight: 14 kDa).
ChIP Use at an assay dependent concentration.
PepArr Use a concentration of 0.2 - 0.02 µg/ml.

Target

  • RelevanceCore component of nucleosome which plays a central role in DNA double strand break (DSB) repair. 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. Post-translational modification for Saccharomyces cerevisiae: Phosphorylated to form H2AS128ph (gamma-H2A) in response to DNA double-strand breaks (DSBs) generated by exogenous genotoxic agents and by stalled replication forks. Phosphorylation is dependent on the DNA damage checkpoint kinases MEC1/ATR and TEL1/ATM, spreads on either side of a detected DSB site and may mark the surrounding chromatin for recruitment of proteins required for DNA damage signaling and repair. Gamma-H2A interacts with ARP4, a shared component of the NuA4 histone acetyltransferase complex and the INO80 and SWR1 chromatin remodeling complexes, and serves to recruit first NuA4, mediating histone H4 acetylation, and subsequently the INO80/SWR1 complexes, facilitating DNA resection, to DSB sites. Gamma-H2A is required for sequestering cohesin around the break site, which is important for efficient post-replicative double-strand break repair by homologous recombination, holding the damaged chromatid close to its undamaged sister template. Gamma-H2A is removed from the DNA prior to the strand invasion-primer extension step of the repair process and subsequently dephosphorylated by PPH3, a component of the histone H2A phosphatase complex (HTP-C). Dephosphorylation is necessary for efficient recovery from the DNA damage checkpoint. N-acetylated by NAT4. Acetylated by ESA1, a component of the NuA4 histone acetyltransferase (HAT) complex, to form H2AK4ac and H2AK7ac. Glutamine methylation at Gln-106 (H2AQ105me) by NOP1 is specifically dedicated to polymerase I. It is present at 35S ribosomal DNA locus and impairs binding of the FACT complex. Sumoylated to from H2AK126su. May lead to transcriptional repression. Post-translational modification for Schizosaccharomyces pombe: Phosphorylated to form H2AS128ph (gamma-H2A) in response to DNA double-strand breaks (DSBs) generated by exogenous genotoxic agents and by stalled replication forks. Phosphorylation is dependent on the DNA damage checkpoint kinases rad3/ATR and tel1/ATM, spreads on either side of a detected DSB site and may mark the surrounding chromatin for recruitment of proteins required for DNA damage signaling and repair. Gamma-H2A is required for recruiting crb2, a modulator of DNA damage checkpoint signaling, to DSB sites. Gamma-H2A is removed from the DNA prior to the strand invasion-primer extension step of the repair process and subsequently dephosphorylated. Dephosphorylation is necessary for efficient recovery from the DNA damage checkpoint. Acetylated by esa1 to form H2AK4ac and H2AK7ac.
  • Cellular localizationNuclear
  • Database links
    • Alternative names
      • H2A1 antibody
      • H2A2 antibody
      • Histone H2A-alpha antibody
      • Histone H2A-beta antibody
      • Histone H2A.1 antibody
      • Histone H2A.2 antibody
      • Hta 1 antibody
      • HTA 2 antibody
      • Hta1 antibody
      • Hta1p antibody
      • HTA2 antibody
      • Hta2p antibody
      • SPT 11 antibody
      • SPT11 antibody
      see all

    Anti-Histone H2A (phospho S129) antibody - ChIP Grade images

    • All lanes : Anti-Histone H2A (phospho S129) antibody - ChIP Grade (ab15083) at 1/500 dilution

      Lane 1 : S.cerevisiae yeast extract (SCYE) + control peptide with Human Histone H2A peptide (ab19751)
      Lane 2 : S.cerevisiae yeast extract with 0.2 % Methyl methanesulfonate (1 hour) with Human Histone H2A peptide (ab19751)
      Lane 3 : SCYE + phospho peptide with Histone H2A peptide - phospho S129 (ab19828)
      Lane 4 : SCYE_M + phospho peptide with Histone H2A peptide - phospho S129 (ab19828)

      Lysates/proteins at 10 µg per lane.


      Performed under reducing conditions.

      Predicted band size : 14 kDa

      The blots were produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membranes were then blocked for an hour. 1 microgram per mL of control- (ab19751, lane 1 and 2) or phospho-peptides (ab19828, lane 3 and 4) were added to the primary antibody ab15083 (rabbit anti-Histone H2A (phospho S129) antibody; diluted 1:500) and loading control ab125247 (mouse anti-GAPDH antibody; diluted 1:20000) and the membranes were incubated with peptide/antibody mixture for 24 hours at 4°C. Antibody binding was detected using infrared (IR)-labelled goat anti-rabbit (green) and  IR-labelled goat anti-mouse (red; insert below) antibodies, diluted 1:20,000, for 1 hour at room temperature before imaging.

    • Serially diluted ab15083 was bound to immobilised phospho (ab19828) - or control (ab19751) peptides (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.

    • All batches of ab15083 are tested in Peptide Array against peptides to different Histone H2A modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H2A - phospho S129 (ab19828), indicating that this antibody specifically recognises the Histone H2A - phospho S129 modification.

      ab19828 - Histone H2A - phospho S129

      ab19751 - Histone H2A - unmodified

    • Anti-Histone H2A (phospho S129) antibody - ChIP Grade (ab15083) at 1 µg/ml + S.cerevisiae (Y190) Whole Cell Lysate at 10 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
      Developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 14 kDa
      Additional bands at : 17 kDa. We are unsure as to the identity of these extra bands.

      Exposure time : 90 seconds


    • Predicted band size : 14 kDa

      Jessica Downs

      Whole cell extracts from wild-type yeast strains were analyzed by 15% SDS-PAGE followed by Western botting using either the affinity purified anti-P-S129 H2A (ab15083) or anti-H2A for a loading control. The MMS treated ells were grown in the presence of 0.10% MMS for 30 min at 30°C prior to lysing.



    • Predicted band size : 14 kDa

      Jessica Downs

      Yeast proteins were prepared by TCA precipitation of whole cell extracts from wild-type yeast in mid-log phase, either treated (lane 1) or not treated (lane 2) with 0.2% MMS for one hr prior to harvesting.

      Extracts were electrophorised on 18% SDS PAGE, transferred to nitrocellulose and blocked for 1 hr in 5% Marvel in TBS-T. The membrane was incubated with ab15083 at 1:1000 in TBS-T for 1 hr at RT, washed and incubated with anti-rabbit secondary (Pierce) for 1 hr at 1:10,000in TBS-T for 1 hr at RT. The blots were visualised with Pierce ECL. 

    References for Anti-Histone H2A (phospho S129) antibody - ChIP Grade (ab15083)

    This product has been referenced in:
    • Felipe-Abrio I  et al. RNA polymerase II contributes to preventing transcription-mediated replication fork stalls. EMBO J 34:236-50 (2015). Read more (PubMed: 25452497) »
    • Wang SH  et al. Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity. PLoS One 10:e0134110 (2015). Read more (PubMed: 26218133) »

    See all 32 Publications for this product

    Product Wall

    Yes, this antibody cross-reacts with Schizosaccharomyces pombe. You can find some references here:

    Carneiro T et al. Telomeres avoid end detection by severing the checkpoint signal transduction pathway. Nature 467:228-32 (2010). ChIP; Schizos...

    Read More

    >We only have full size vials of our products, so unfortunately we do not have a sample to send out. The antibody is fully guaranteed to work in Western blot and ChIP with S. cerevisiae samples for up to 612months after purchase. If you are interested ...

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Saccharomyces cerevisiae Cell lysate - whole cell (BY4741 wild-type cells)
    Loading amount 2e+007 cells
    Specification BY4741 wild-type cells
    Treatment 0, 1 and 10 µM nitrogen mustard for 2h
    Gel Running Conditions Reduced Denaturing (12%)
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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    Verified customer

    Submitted Apr 18 2012

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