Recombinant
RabMAb

Recombinant Anti-Histone H2A.X antibody [EPR895] - BSA and Azide free (ab217838)

Overview

  • Product name

    Anti-Histone H2A.X antibody [EPR895] - BSA and Azide free
    See all Histone H2A.X primary antibodies
  • Description

    Rabbit monoclonal [EPR895] to Histone H2A.X - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ChIP, ICC/IF, IP, Flow Cytmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Histone H2A.X aa 1-100. The exact sequence is proprietary.

  • General notes

    Ab217838 is the carrier-free version of ab124781. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    ab217838 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    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 ab217838 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 Use at an assay dependent concentration. Detects a band of approximately 14 kDa (predicted molecular weight: 15 kDa).
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ChIP Use at an assay dependent concentration. PubMed: 22275873
ICC/IF Use at an assay dependent concentration.

ab217838 does not work on PFA fixed cells but does work on methanol fixed cells.

IP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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

  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H2A.X with purified ab124781 at 1/100 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Histone H2A.X with purified ab124781 at a dilution of 1/1000. Cells were fixed with either 4% PFA (top) or 100% methanol (bottom) and permeabilized with 0.1% TritonX-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.

    ab7291 mouse anti-Tubulin (1/1000) followed by ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were used to label tubulin. DAPI was used as the nulear counterstain.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Histone H2A.X with unpurified ab124781 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

  • Histone H2A.X was immunoprecipitated using 5ug of ab124781 from 200ul of HeLa whole cell extract lysate diluted to 0.5mg/ml in RIPA and 50ul of Protein G magnetic beads. No antibody was added to the control (-).

    The antibody was incubated under agitation with the Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C. 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab124781 at 1ug/ml. The Secondary antibody was Mouse monoclonal SB62a Anti-Rabbit IgG light chain (HRP) (ab99697) at 1/10,000 dilution.

    Band: 15kDa; Histone H2A.X

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124781).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labeling Histone H2A.X with purified ab124781 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol and sodium azide (ab124781).

References

ab217838 has not yet been referenced specifically in any publications.

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