Anti-Histone H2A.X antibody - ChIP Grade (ab11175)
- Product nameAnti-Histone H2A.X antibody - ChIP GradeSee all Histone H2A.X primary antibodies ...
- DescriptionRabbit polyclonal to Histone H2A.X - ChIP Grade
- Tested applicationsIHC-P, WB, IP, ChIP, ICC/IF, IHC-Fr more details
- Species reactivityReacts with: Mouse, Rat, Human, Monkey
Predicted to work with: Rabbit, Rhesus monkey, Gorilla
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.
- Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
- Storage bufferPreservative: 0.1% Sodium Azide
Constituents: 150mM Tris, 60mM Citrate, 8mM Phosphate, pH 7-8
- Concentration information loading...
- PurityImmunogen affinity purified
- Purification notesAntibodies were affinity purified using the peptide immobilized on solid support.
- Clonality Polyclonal
- Research Areas
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.
|IHC-P||IHC-P: Use at an assay dependent concentration.|
|WB||WB: 1/5000 - 1/15000. Detects a band of approximately 15 kDa.|
|IP||IP: Use at 5-20 µg/mg of lysate.|
|ChIP||ChIP: Use at an assay dependent dilution. PubMed: 19380460|
|ICC/IF||ICC/IF: 1/2500. (see review). Use periodate-lysine-PFA fixative.|
- 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 antibody - ChIP Grade images
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.
Asynchronous HeLa cells were paraformaldehyde fixed (4%), immunofluorescently labeled with anti-H2AX (ab11175) and counterstained with DAPI. Merged images present the DAPI and H2A.X channel information as red and green, respectively. Scale bar represents 20
ab11175 appears to bind to a nuclear location. Nuclear binding appears to be reduced upon pre-incubation with the phosphorylated blocking peptide, ab15645.
Anti-Histone H2A.X antibody - ChIP Grade (ab11175) at 1/2000 dilution + Rat C6 Cells, whole cell lysate at 25 µg
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
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.
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:
2) Histone H2A.X;
References for Anti-Histone H2A.X antibody - ChIP Grade (ab11175)
This product has been referenced in:
- Soldi M & Bonaldi T The proteomic investigation of chromatin functional domains reveals novel synergisms among distinct heterochromatin components. Mol Cell Proteomics 12:764-80 (2013). WB, ChIP . Read more (PubMed: 23319141) »
- Castro-Vega LJ et al. Telomere crisis in kidney epithelial cells promotes the acquisition of a microRNA signature retrieved in aggressive renal cell carcinomas. Carcinogenesis N/A:N/A (2013). Human . Read more (PubMed: 23358853) »