Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)

Overview

• Product nameAnti-gamma H2A.X (phospho S139) antibody [9F3]See all gamma H2A.X primary antibodies ...
• Description
  Mouse monoclonal [9F3] to gamma H2A.X (phospho S139)
• Tested applicationsFlow Cyt, WB, IP, ICC/IF, IHC-P, In Situ Hybridisation more details
• Species reactivity
  Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Monkey
  
• Immunogen

  Synthetic peptide of phosphorylated (Ser139) human Histone H2A.X.

• Positive controlHeLa heat shocked cell lysate for Western blot.

Properties

• FormLiquid
• Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C long term. Avoid repeated freeze / thaw cycles.
• Storage bufferPreservative: 0.09% Sodium Azide
  Constituents: 50% Glycerol, PBS, pH 7.2
• Concentration information loading...
• PurityProtein G purified
• Purification notesThis antibody was purified using affinity chromatography.
• Clonality Monoclonal
• Clone number9F3
• IsotypeIgG
• Research Areas
  • Epigenetics and Nuclear Signaling
  • Histones
  • Variants

  • Epigenetics and Nuclear Signaling
  • Histones
  • H2A
  • Unmodified

  • Epigenetics and Nuclear Signaling
  • DNA / RNA
  • DNA Damage & Repair
  • DNA Damage Response
  • DNA Damage Recognition

Applications

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
  modificationsPhosphorylated 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.

Target information above from: UniProt accession P16104 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
• Database links
  • Entrez Gene: 3014 Human
  • Entrez Gene: 15270 Mouse
  • Entrez Gene: 500987 Rat
  • Omim: 601772 Human
  • SwissProt: P16104 Human
  • SwissProt: P27661 Mouse
  • Unigene: 477879 Human
  • Unigene: 245931 Mouse

  • Unigene: 2850 Rat

  see all

• Alternative names
  H2A histone family member X antibodyH2A.FX antibodyH2A.X antibody

  H2A/X antibodyH2AFX antibodyH2AX antibodyH2AX_HUMAN antibodyHistone H2A.x antibody

  see all

Anti-gamma H2A.X (phospho S139) antibody [9F3] images

• 

  Western blot - gamma H2A.X (phospho S139) antibody [9F3] (ab26350)
  All lanes : Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350) at 1/1000 dilution
  
  Lane 1 : Untreated Zebrafish embryos.
  Lane 2 : Zebrafish embryos treated with 25 Gy of X-Ray.
  Lane 3 : Zebrafish embryos treated with 50 Gy of X-Ray
  
  Secondary
  Goat anti-mouse HRP at 1/10000 dilution

  This image is courtesy of an anonymous Abreview

  See Abreview

• 

  Flow Cytometry - gamma H2A.X (phospho S139) antibody [9F3] (ab26350)

  Overlay histogram showing HeLa cells stained with ab26350 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab26350, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was a mix of mouse IgG1 [ICIGG1], (ab91353, 1μg/1x10⁶ cells), IgG2a [ICIGG2A], (ab91361, 1μg/1x10⁶ cells), IgG2b [PLPV219], (ab91366, 1μg/1x10⁶ cells), IgG3 [MG3-35], (ab18394, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  
  

• 

  Immunocytochemistry/ Immunofluorescence - gamma H2A.X (phospho S139) antibody [9F3] (ab26350)This image is a courtesy of Pinton Alain

  ab26350 staining gamma H2A.X in pig spermatocytes by Immunocytochemistry/ Immunofluorescence. Cells were fixed with formaldehyde, permeabilized with Triton x100 and blocking with 0.15% BSA was performed for 30 minutes at 25⁰C was performed. Samples were incubated with primary antibody (1/100: in PBS + 0.15% BSA+0.1% Tween 20) for 12 hours at 25°C. An Alexa Fluor^®488-conjugated goat monoclonal to mouse IgG was used at dilution at 1/100 as secondary antibody.

  See Abreview

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)
  ab26350 (1µg/ml) staining gamma H2A in human spleen, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining .
  Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  
• 

  Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
  ab26350 (1/500) staining gamma H2A.X (phospho S139) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

  See Abreview

• 

  Western blot - gamma H2A.X (phospho S139) antibody [9F3] (ab26350)
  All lanes : Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)
  
  Lane 1 : Molecular weight marker
  Lane 2 : Cell lysates prepared from human Jurkat cells
  Lane 3 : Cell lysates prepared from human Jurkat cells treated with staurosporine
  Lane 4 : Cell lysates prepared from mouse NIH3T3 cells
  Lane 5 : Cell lysates prepared from CHO-K1 cells
  Lane 6 : Cell lysates prepared from Rat-2 cells