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

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
  • Tested applicationsIHC-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

    Synthetic peptide, which represents a portion of the C-terminus of human histone H2AX (LocusLink ID 3014).

  • Positive control
    • Tested with human HEK293, human G-361 and mouse embryonic fibroblast cells.
  • General notesThe 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

  • 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 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.

  • 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.

  • These images were kindly supplied as part of the review submitted by Geza-Fejes-Toth.
  • Anti-Histone H2A.X antibody - ChIP Grade (ab11175) at 1/2000 dilution + Rat C6 Cells, whole cell lysate at 25 µg

    Secondary
    HRP conjugated goat anti-rabbit antibody at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Observed band size : 16 kDa (why is the actual band size different from the predicted?)


    Exposure time : 1 second

    This image is courtesy of an anonymous Abreview

    See Abreview

  • 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 murine aorta by Immunohistochemistry (Frozen sections).
    Tissue was fixed in acetone, permeabilized using 0.3% Triton, blocked with 10% serum for 30 minutes at 22°C and then incubated with ab11175 at a 1/1000 dilution for 16 hours at 4°C. The secondary used was ab6793, sheep polyclonal secondary antibody to rabbit IgG - H&L (TR), used at a 1/1000 dilution.

    See Abreview

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

This product has been referenced in:
  • Huang SC  et al. Tumor necrosis factor suppresses NR5A2 activity and intestinal glucocorticoid synthesis to sustain chronic colitis. Sci Signal 7:ra20 (2014). Read more (PubMed: 24570488) »
  • Leung JW  et al. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling. PLoS Genet 10:e1004178 (2014). WB ; Human . Read more (PubMed: 24603765) »

See all 27 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Aorta)
Specification Aorta
Fixative Acetone
Permeabilization Yes - 0.3% Triton
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Dec 20 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (vascular smooth muscle cells)
Loading amount 100000 cells
Specification vascular smooth muscle cells
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted May 14 2010

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
Sample Human Cell (T98G Human brain glioblastoma)
Specification T98G Human brain glioblastoma
Permeabilization Yes - 0.1% v/v Triton X-100 pH 7.4 for 5 min at RT
Fixative Paraformaldehyde
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Dr. Dimitra Kalamida

Verified customer

Submitted Dec 06 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Rat Cell lysate - nuclear (vascular smooth muscle cells)
Specification vascular smooth muscle cells
Treatment 80 uM tert-butyl BHP
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jun 24 2013

Thank you for contacting us.
We work with one of our collaborators to produce this antibody who in the past has suggested storing it at 4ºC. However, we have recently made the decision to store it at -20ºC rather than 4ºC. This is because we have ...

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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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Thank you for contacting us.
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.
I hope this...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (testis)
Specification testis
Fixative Paraformaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer
Permeabilization Yes - 0.1%Triton X100
Blocking step BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 37°C
Username

Abcam user community

Verified customer

Submitted May 01 2012

Thank you for calling us to update the progress you have made in using ab11175 in western blotting. I am sorry that the results have not improved. From what you have described, it seems that it may be related to the antibody itself, as you have perform...

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Thank you for your call yesterday and for sending these images. They are very helpful to better understand the situation.

The most likely cause of this problem is cross-reaction of the primary antibody with the blocking buffer. I would recomm...

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