Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914)

Overview

  • Product name

    Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade
    See all Histone H2A.X primary antibodies
  • Description

    Rabbit monoclonal [EPR22820-23] to Histone H2A.X - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: PepArr, ChIP, ICC/IF, Flow Cyt, WB, IHC-P, IPmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human Histone H2A.X aa 100 to the C-terminus. The exact sequence is proprietary.
    Database link: P16104

  • Positive control

    • WB: Wild-type HAP1 whole, Human brain, HeLa, 293T and HEK-293 lysates. IHC-P: Human breast carcinoma and Human testis tissues. ICC/IF: HeLa cells. FC: HeLa cells. IP: HeLa cells.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab229914 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
PepArr Use a concentration of 0.1 µg/ml.

 

 

ChIP Use 5 µg for 25 µg of chromatin.

 

 

ICC/IF 1/100.

 

 

Flow Cyt 1/50.

 

 

WB 1/1000. Predicted molecular weight: 15 kDa.

 

 

IHC-P 1/200.

False

 

 

IP 1/30.

 

 

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

  • All lanes : Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution

    All lanes : Human brain tissue

    Lysates/proteins at 20 µg per lane.

    Secondary
    Lane 1 : VeriBlot for IP secondary antibody (HRP) at 1/1000 dilution
    Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 15 kDa
    Observed band size: 16,25 kDa
    why is the actual band size different from the predicted?



    This blot was developed using a higher sensitivity ECL substrate.

  • Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Histone H2A.X with ab229914 at 1/200 dilution (2.56 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human testis (PMID/ 24059746). The section was incubated with ab229914 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101)

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H2A.X with ab229914 at 1/100 dilution, followed by Ab229914 anti- Histone H2A.X ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line is observed. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Histone H2A.X with ab229914 at 1/50 dilution(Red), compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.

    The ChIP was performed with 25 µg of chromatin, 5 µg of ab229914 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20µl of Protein A/G sepharose beads.  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.

    *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol.

  • All batches of ab229914 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).

    Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

    The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

  • Histone H2A.X was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab229914 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab229914 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

    Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

    Lane 2: ab229914 IP in HeLa whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229914 in HeLa whole cell lysate

    Blocking and dilution buffer and concentration/ 5% NFDM/TBST.

    Exposure time/ 30 seconds.

     

  • Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution + HEK-293 (human embryonic kidney epithelial cell), histone extract at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 15 kDa
    Observed band size: 16, 25 kDa why is the actual band size different from the predicted?



    A 25-kDa band, likely to be ubiquitinated H2A.X, is observed. The molecular weights are consistent with what have been described in the literature (PMID: 24603765)

  • All lanes : Anti-Histone H2A.X antibody [EPR22820-23] - ChIP Grade (ab229914) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Histone H2A.X knockout HAP1 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 15 kDa
    Observed band size: 16 kDa why is the actual band size different from the predicted?



    Ab229914 was shown to specifically react with Histone H2A.X in wild-type HAP1 cells as signal was lost in Histone H2A.X knockout cells. Wild-type and Histone H2A.X knockout samples were subjected to SDS-PAGE. ab229914 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.   The blot was developed on a BIO-RAD® ChemiDocâ„¢ MP instrument using the ECL technique. Blocking/Diluting buffer and concentration: 5% NFDM/TBST  Exposure Time: 37 seconds

  • Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling Histone H2A.X with ab229914 at 1/200 dilution (2.56 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human breast carcinoma (PMID/ 27006338). The section was incubated with ab229914 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

    Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101)

    .

    Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

References

ab229914 has not yet been referenced specifically in any publications.

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