Anti-Histone H2A.X (phospho S139) [EP854(2)Y] antibody - ChIP Grade (ab81299)
- Product nameAnti-Histone H2A.X (phospho S139) [EP854(2)Y] antibody - ChIP GradeSee all Histone H2A.X primary antibodies ...
- DescriptionRabbit monoclonal [EP854(2)Y] to Histone H2A.X (phospho S139) - ChIP Grade
- Tested applicationsWB, IHC-P, ICC/IF, CHIPseq, Flow Cyt more details
- Species reactivityReacts with: Mouse, Rat, Human
A synthetic phospho-peptide corresponding to residues surrounding serine 139 of Human Histone H2A.X protein was used as the immunogen.
- Positive control
- Jurkat cell lysate and Human kidney transitional cell carcinoma.
- General notes
Produced under U.S. Patent No. 5,675,063.
This product is available conjugated to DyLight® 488 see ab126708.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%
- PurityTissue culture supernatant
- Clonality Monoclonal
- Clone numberEP854(2)Y
- Research Areas
Our Abpromise guarantee covers the use of ab81299 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||WB: 1/100000 - 1/500000. Predicted molecular weight: 15 kDa.|
|IHC-P||IHC-P: 1/100 - 1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|ICC/IF||ICC/IF: 1/50 - 1/100.|
|CHIPseq||CHIPseq: Use at an assay dependent concentration. PubMed: 20714222|
|Flow Cyt||Flow Cyt: 1/100.|
- 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.
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.
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Anti-Histone H2A.X (phospho S139) [EP854(2)Y] antibody - ChIP Grade images
Overlay histogram showing HeLa cells stained with ab81299 (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 (ab81299, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
IHC-P image of γH2A.X staining on Mouse testis sections using ab81299 (1:5000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab81299 was diluted 1:5000 in TBS buffer (containing BSA and Azide) and sections were then incubated with ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).
IHC-P image of γH2A.X staining on Rat testis sections using ab81299 (1:5000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab81299 was diluted 1:5000 in TBS buffer (containing BSA and Azide) and sections were then incubated with ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).
All lanes : Anti-Histone H2A.X (phospho S139) [EP854(2)Y] antibody - ChIP Grade (ab81299) at 1/500000 dilution
Lane 1 : Jurkat cell lysates Jurkat cell lysates
Lane 2 : Jurkat cell lysates treated with etoposide
Lysates/proteins at 10 µg per lane.
HRP Labelled goat anti-rabbit at 1/2000 dilution
Predicted band size : 15 kDa
Observed band size : 15 kDa
ab81299, at 1/100 staining Human kidney transitional cell carcinoma by Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney transitional cell carcinoma using 1/100 ab81299.
ICC/IF image of ab81299 stained HepG2 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 (ab81299, neat) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) 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.
References for Anti-Histone H2A.X (phospho S139) [EP854(2)Y] antibody - ChIP Grade (ab81299)
This product has been referenced in:
- Wartlick F et al. DNA damage response (DDR) induced by topoisomerase II poisons requires nuclear function of the small GTPase Rac. Biochim Biophys Acta 1833:3093-3103 (2013). Human . Read more (PubMed: 23999236) »
- Geuting V et al. ATM release at resected double-strand breaks provides heterochromatin reconstitution to facilitate homologous recombination. PLoS Genet 9:e1003667 (2013). ICC/IF ; Human . Read more (PubMed: 23935532) »