Thermo Fisher Scientific Phospho-Histone H2A.X (Ser139) Monoclonal Antibody (CR55T33), eFluor 660, eBioscience

Applications

Tested Dilution

Publications

Western Blot (WB)

1:400

View 2 publications 2 publications

Immunohistochemistry (IHC)

-

View 1 publication 1 publication

Immunocytochemistry (ICC/IF)

1:50

-

Flow Cytometry (Flow)

5 µL (0.125 µg)/test

View 3 publications 3 publications

Product Specifications

Published species

Human

Host/Isotype

Mouse / IgG1, kappa

Recommended Isotype Control

Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), eFluor™ 660, eBioscience™

Class

Monoclonal

Type

Antibody

Clone

CR55T33

Conjugate

eFluor™ 660 eFluor™ 660 eFluor™ 660

View additional formats

• Unconjugated
• Alexa Fluor 488
• eFluor 570
• PE
• PerCP-eFluor 710
• Request custom conjugation

Excitation/Emission Max

651/668 nm View spectra

Form

Liquid

Concentration

5 µL/Test

Storage conditions

4° C, store in dark, DO NOT FREEZE!

Shipping conditions

Wet ice

RRID

AB_2574398

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, and immunocytochemistry.

Applications Tested: This CR55T33 antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of treated human peripheral blood cells using the Foxp3/Transcription Factor Buffer Set (Product # 00-5523-00) and protocol. This can be used at 5 µL (0.125 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. The CR55T33 antibody has also been tested by immunocytochemistry of methanol-fixed human cells and can be used at less than or equal to 10 µg/mL. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

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.

Excitation: 633-647 nm; Emission: 668 nm; Laser: Red Laser.

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.