Product: Histone H2A.X Antibody
Catalog: AF6187
Description: Rabbit polyclonal antibody to Histone H2A.X
Application: WB IHC
Reactivity: Human, Mouse, Rat, Monkey
Prediction: Bovine, Sheep, Dog
Mol.Wt.: 15kDa; 15kD(Calculated).
Uniprot: P16104
RRID: AB_2835070

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 100ul $280 In stock
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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:200
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse,Rat,Monkey
Prediction:
Bovine(91%), Sheep(91%), Dog(91%)
Clonality:
Polyclonal
Specificity:
Histone H2A.X Antibody detects endogenous levels of total Histone H2A.X.
RRID:
AB_2835070
Cite Format: Affinity Biosciences Cat# AF6187, RRID:AB_2835070.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

AW228881; H2A histone family member X; H2A.FX; H2A.X; H2a/x; H2AFX; H2AX; H2AX histone; H2AX_HUMAN; Hist5.2ax; Histone 2A; Histone 2AX; Histone H2A.X; Histone H2AX; RGD1566119;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Description:
Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes.
Sequence:
MSGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGGVTIAQGGVLPNIQAVLLPKKTSATVGPKAPSGGKKATQASQEY

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Bovine
91
Sheep
91
Dog
91
Pig
0
Horse
0
Xenopus
0
Zebrafish
0
Chicken
0
Rabbit
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - P16104 As Substrate

Site PTM Type Enzyme
S2 Phosphorylation
K6 Acetylation
K10 Acetylation
K14 Ubiquitination
K16 Ubiquitination
S19 Phosphorylation
K119 Acetylation
K119 Ubiquitination
K120 Ubiquitination
T121 Phosphorylation
S122 Phosphorylation
T124 Phosphorylation
K134 Methylation
K134 Sumoylation
K134 Ubiquitination
T137 Phosphorylation
S140 Phosphorylation Q13315 (ATM) , P45984 (MAPK9) , P45983 (MAPK8)
Y143 Phosphorylation Q9UIG0 (BAZ1B)

Research Backgrounds

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.

PTMs:

Phosphorylated on Ser-140 (to form gamma-H2AX 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 (By similarity). 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. Ubiquitination at Lys-14 and Lys-16 (H2AK13Ub and H2AK15Ub, respectively) in response to DNA damage is initiated by RNF168 that mediates monoubiquitination at these 2 sites, and 'Lys-63'-linked ubiquitin are then conjugated to monoubiquitin; RNF8 is able to extend 'Lys-63'-linked ubiquitin chains in vitro. H2AK119Ub and ionizing radiation-induced 'Lys-63'-linked ubiquitination (H2AK13Ub and H2AK15Ub) are distinct events.

Acetylation at Lys-37 increases in S and G2 phases. This modification has been proposed to play a role in DNA double-strand break repair (By similarity).

Subcellular Location:

Nucleus. Chromosome.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Subunit Structure:

The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA (Probable). Interacts with numerous proteins required for DNA damage signaling and repair when phosphorylated on Ser-140. These include MDC1, TP53BP1, BRCA1 and the MRN complex, composed of MRE11, RAD50, and NBN. Interaction with the MRN complex is mediated at least in part by NBN. Also interacts with DHX9/NDHII when phosphorylated on Ser-140 and MCPH1 when phosphorylated at Ser-140 or Tyr-143. Interacts with ARRB2; the interaction is detected in the nucleus upon OR1D2 stimulation. Interacts with WRAP53/TCAB1.

(Microbial infection) Interacts with Epstein-Barr virus protein EBNA6.

Family&Domains:

The [ST]-Q motif constitutes a recognition sequence for kinases from the PI3/PI4-kinase family.

Belongs to the histone H2A family.

Research Fields

· Cellular Processes > Cell growth and death > Necroptosis.   (View pathway)

· Human Diseases > Substance dependence > Alcoholism.

· Human Diseases > Immune diseases > Systemic lupus erythematosus.

References

1). Li et al. Salvianolic acid B suppresses hepatic fibrosis by inhibiting ceramide glucosyltransferase in hepatic stellate cells. Acta Pharmacologica Sinica 2023 Jan 10. (PubMed: 36627345) [IF=8.2]

2). Zhou Q et al. PolyG mitigates silica-induced pulmonary fibrosis by inhibiting nucleolin and regulating DNA damage repair pathway. BIOMEDICINE & PHARMACOTHERAPY 2020 Feb 6;125:109953 (PubMed: 32036217) [IF=7.5]

Application: WB    Species: mouse    Sample: lung

Fig. 2.| Measure the effect of PolyG on silica-induced DNA damage and nucleolin expression in a mouse model of silicosis. (A) expression of nucleolin and DSBs marker γ-H2AX in mouse lung tissue as measured by Western blotting.

3). Zhang T et al. Insight into the Practical Models for Prediciting the Essential Role of the Cytochrome P450-mediated Biotransformation in Emodin-associated Hepatotoxicity. TOXICOLOGY 2021 Sep 4;462:152930. (PubMed: 34492313) [IF=4.5]

Application: WB    Species: Mice    Sample: THLE-2 cells

Fig. 3. Both electrophility and the ROS producing activity of emodin contributed to its toxicity in THLE-2 cells. (A) Effect of emodin on ROS generation in THLE-2 cells. Cells were treated with indicated concentra- tions of emodin for 24 h, and intracelluar ROS was monitored with DCFH-DA probe. Data were presented as means ± SD. *P < 0.05, **P < 0.01, compared with control. (B) Effect of catalase on emodin-induced cell toxicity in THLE-2 cells. Cells were treated with emodin in the presence or absence of catalase (17.5 IU/ mL) for 24 h. Cell viability was tested by MTT assay. Data were presented as means ± SD. **P < 0.01, ***P < 0.001, compared with the cor- responding control; ### P < 0.001, emodin and catalase cotreatment group vs. emodin treat- ment group. (C) Effect of emodin on protein thiol (protein-SH) levels in THLE-2 cells. Cells were treated with indicated concentrations of emodin for 24 h. Intracellular levels of protein- SH were measured. Data were presented as means ± SD. *P < 0.05, ***P < 0.001, compared with control. (D) Effect of emodin on the status of H 2 AX activation in THLE-2 cells. Cells were treated with emodin for 24 h, and equal amounts of total cell lysates (50 μ g) were loaded and subjected to immunoblot analysis. Data represent the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001, compared with control group.

4). Qi et al. Betulin attenuates pneumolysin‐induced cell injury and DNA damage. Journal of Applied Microbiology 2020 Jul 4. (PubMed: 32621771) [IF=4.0]

Application: IF/ICC    Species: Human    Sample: A549 cells

Fig 4. Betulin inhibits PLY-induced DNA damage. The A549 cells were exposed in vitro to PLY with or without betulin for 6 h. (A) The images were selected from immunofluorescence assays and were captured by confocal microscopy. (B) Histogram of the γH2AX-positive cell ratio from at least 100 cells for each sample. γH2AX-positive cells (≥3 foci per nucleus) were quantified as the percentage of positive cells. ** indicates P < 0.01 compared to the betulin-free group according to one-way ANOVA followed by LSD tests (n=3).

5). Yanjuan He et al. Chidamide, a subtype-selective histone deacetylase inhibitor, enhances Bortezomib effects in multiple myeloma therapy. Journal of Cancer 2021 Aug 27;12(20):6198-6208. (PubMed: 34539893) [IF=3.9]

Application: WB    Species: Human    Sample: MM cells

Figure 4 A combination of Chidamide and Bortezomib increases production of ROS dependent DNA damage and the changes of cell apoptosis and cycle pathway in MM cells. (A) ARP-1 cells were pretreated with or without NAC (15.0 mmol/L) for 2 hours at 37°C and then incubated with CHI (1.0 µmol/L) and/or BTZ (5.0 nmol/L) for 24 hours, then ROS generation was detected. (B) ARP-1 cells were pretreated with or without 15 mmol/L NAC and then treated with Chidamide or Bortezomib alone or in combination, and cell viabilities were evaluated using CCK-8 assays. (C) The expression of γ-H2AX in ARP-1 cells treated with CHI (1.0 µmol/L) and/or BTZ (5.0 nmol/L) were determined by Western blot. (D) Representative images of γ-H2AX (Red) and nuclei (Blue) in ARP-1 cells treated with single agent or combination for 24 hours by immunofluorescence assay. Scale bars represent 20 µm. (E, F) Western blot analysis of the expressions of cleaved caspase3, cleaved caspase8, cleaved PARP-1 and HDAC1 in XG1 (E) and ARP-1 (F) cells after 48 hours treatment with single agent or in combination. Error bars indicate mean ± SD. **p < 0.01.

6). Inhibition of poly (ADP-ribose) Polymerase-1 (PARP-1) improves endothelial function in pulmonary hypertension. Pulmonary Pharmacology & Therapeutics [IF=3.2]

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