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PRODUCT CODE: ER1901-70

Histone H2A.x Rabbit Polyclonal Antibody (ER1901-70)

Applications

  • WB

  • IHC-P

  • ICC

  • IF

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

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Western blot analysis of Histone H2A.x on MCF-7 cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-70, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of Histone H2A.x on MCF-7 cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-70, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • ICC staining of Histone H2A.x in SHSY5Y cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ER1901-70, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
  • Immunofluorescence analysis of paraffin-embedded rat brain using anti-Histone H2A.x antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature , washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-70, 1/100) at 4 ° C overnight. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue)。
  • Immunohistochemical analysis of paraffin-embedded Rat testicular tissue using anti-Histone H2A.x antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-70, 1/100) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human lung cancer tissue using anti-Histone H2A.x antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-70, 1/100) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-Histone H2A.x antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-70, 1/100) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded mouse fallopian tube tissue using anti-Histone H2A.x antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ER1901-70, 1/100) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
  • Flow cytometric analysis of Histone H2A.x was done on SHSY5Y cells. The cells were fixed, permeabilized and stained with the primary antibody (ER1901-70, 1/100) (purple). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated goat anti-rabbit IgG Secondary antibody at 1/500 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; yellow).
Western blot analysis of Histone H2A.x on MCF-7 cell lysate. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ER1901-70, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.

Applications

  • WB

  • IHC-P

  • ICC

  • IF

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Rabbit polyclonal primary

Product Name

Histone H2A.x Rabbit Polyclonal Antibody (ER1901-70)

Immunogen

Synthetic peptide within human histone h2ax aa 1-50 / 143.

Host

Rabbit

Positive Control

MCF-7 cell lysates, SHSY5Y, Rat testicular tissue, human lung cancer tissue, human liver tissue, mouse fallopian tube tissue.

Conjugation

Unconjugated

Clonality

Polyclonal

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

1 ug/ul

PURIFICATION

Peptide affinity purified.

MOLECULAR WEIGHT

15 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB:1:500-1:1000

  • IHC-P:1:100-1:500

  • ICC/IF:1:100-1:500

  • FC:1:50-1:100

TARGET

UNIPROT #

P16104

PROTEIN NAME

Histone H2A.x

SYNONYMS

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; Histone H2AX antibody; RGD1566119 antibody

SEQUENCE SIMILARITIES

Belongs to the histone H2A family.

DEVELOPMENTAL STAGE

Synthesized in G1 as well as in S-phase.

POST-TRANSLATIONAL MODIFICATION

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

FUNCTION

Histone H2A.X is a member of the Histone H2A family, which is involved in nucleosomal organization of chromatin. The H2AFX gene is located in close proximity to the Porphobilinogen deaminase (PBG-D) gene in both mouse and human, and maps to chromosome 9 and 11q23, respectively. H2A.X differs from the other members of the H2A family by the presence of a highly conserved C-terminal motif. It is rapidly phosphorylated in response to ionizing radiation and plays an important role in the recognition and repair of DNA double stranded breaks. The phosphorylated form of H2A.X, designated γ-H2A.X, forms nuclear foci at the heavy chain constant region of cells involved in class switch recombination (CSR), a region-specific DNA reaction that replaces one immunoglobulin heavy chain constant region gene with another. The phosphorylated γ-H2A.X is also thought to initiate subsequent repair factors, including Rad50, Rad51 and BRCA1.