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Invitrogen
Phospho-Histone H2A.X (Ser139) Monoclonal Antibody (CR55T33), eBioscience™
Application
FIGURE: 1 / 11
Phospho-Histone H2A.X (Ser139) Antibody (14-9865-80) in ICC/IF
Immunofluorescence analysis of phospho-H2A.X was performed using 70% confluent log phase HeLa cells treated with hydroxyurea (3 mM for 6 hr). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Phospho-Histone H2A.X (Ser139) Monoclonal Antibody (CR55T33), eB... View More
Product Details
14-9865-80
Applications
Tested Dilution
Publications
Product Specifications
Species Reactivity
Human,
Mouse
Published species
Human
Host/Isotype
Mouse
/ IgG1, kappa
Class
Monoclonal
Type
Antibody
Clone
CR55T33
Conjugate
Unconjugated
Form
Liquid
Concentration
0.5 mg/mL
Purification
Affinity chromatography
Storage buffer
PBS, pH 7.2
Contains
0.09% sodium azide
Storage conditions
4°C
Shipping conditions
Ambient (domestic); Wet ice (international)
RRID
AB_2573047
Product Specific Information
Description: The CR55T33 monoclonal antibody recognizes phosphorylated serine 139 of human and mouse H2AX. H2AX is a member of the H2A histone family that complex with DNA and other histones to form the repeating nucleosome units characteristic of eukaryotic chromatin. Nucleosomes consist of approximately 147 base pairs of DNA wrapped around an octamer of histones composed of two each of the four histone proteins: H2A, H2B, H3 and H4. After induction of DNA damage such as double-strand breaks by irradiation, genotoxic stresses, replication errors or gene recombination, PI3K-like kinases (e.g., ataxia telangiectasia mutated (ATM), ataxia telangiectasia Rad-3-related (ATR), and DNA-dependent protein kinase (DNA-PK) are activated to phosphorylate serine 139 in H2AX. This early phosphorylation event plays a critical role in recruiting proteins involved in DNA repair.
The monoclonal antibody CR55T33 recognizes a single band of approximately 15 kDa on reduced cell lysates from Jurkat cells stimulated with etoposide.
Applications Reported: This CR55T33 antibody has been reported for use in intracellular staining followed by flow cytometric analysis, western blotting, immunohistochemical staining of formalin-fixed paraffin embedded tissue sections, and immunocytochemistry (fluorochrome-conjugated CR55T33 is recommended for use in intracellular flow cytometry).
Applications Tested: The CR55T33 has been tested by immunocytochemistry of methanol-fixed cells and by immunohistochemistry of human tissue using either low or high pH antigen retrieval and can be used at 5 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. This CR55T33 antibody has also been tested by intracellular staining and flow cytometric analysis of stimulated Jurkat cells using the Foxp3/Transcription Factor Buffer Set (Product # 00-5523-00) and protocol. This can be used at 0.03 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.
Protocols: We recommend Protocol B: One-step protocol: intracellular (nuclear) proteins. Alternatively, Protocol C: Two-step protocol: Fixation/Methanol can also be used. Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins cannot be used. All Protocols can be found in the "Staining intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.
eFluor® 660 is a replacement for Alexa Fluor® 647. eFluor® 660 emits at 659 nm and is excited with the red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochrome.
Purity: Greater than 90%, as determined by SDS-PAGE.
Aggregation: Less than 10%, as determined by HPLC.
Filtration: 0.2 µm post-manufacturing filtered.
Target Information
Histone H2A.X (H2AX) is a member of the histone H2A family which is one of the four core histones making up the nucleosome core particle. In eukaryotes, DNA double strand breaks (DSBs) have been shown to trigger the phosphorylation of serine 139 at the carboxy terminus of histone H2AX resulting in gamma-H2AX. The phosphorylation of H2AX can be detected by Western blotting or immunofluorescence, revealing the frequency of DSBs. The phosphatidylinositol 3-kinases have been implicated in H2AX phosphorylation, but it is unclear if ATM is the primary H2AX kinase or if other members of the family such as DNA-PK and ATR contribute in a similar manner. Structurally, H2A.x contains 143 amino acid residues. Histone H2A.X is considered a basal histone, being synthesized in G1 as well as in S-phase, and its mRNA contains polyA addition motifs and a polyA tail along with the conserved stem-loop and U7 binding sequences involved in the processing and stability of replication type histone mRNAs. There are two forms of Histone H2A.X mRNA, one about 1600 bases long and contains polyA; the other about 575 bases long, lacking polyA. The short form behaves as a replication type histone mRNA, while the longer behaves as a basal type histone mRNA. Histone H2A.X maps to the 11q23.2-q23.3 region of the human chromosome. Histone H2A.x contributes to histone-formation and therefore the structure of DNA. Histone H2A variant H2A.x specifically regulates the interaction of MDC1 (mediator of DNA damage checkpoint protein 1), a DNA repair protein to the sites of DNA damage.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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Bioinformatics
Protein Aliases: gamma H2AX; H2A histone family, member X; H2a/x; H2AX histone; histone 5 protein 2ax; Histone H2A.X; Histone H2a/x; Histone H2AX
Gene Aliases: AW228881; gammaH2ax; H2A.X; H2A/X; H2AFX; H2AX; Hist5-2ax
UniProt ID: (Human) P16104, (Mouse) P27661
Entrez Gene ID: (Human) 3014, (Mouse) 15270
Performance Guarantee
If an Invitrogen™ antibody doesn't perform as described on our website or datasheet,we'll replace the product at no cost to you, or provide you with a credit for a future purchase.*
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