Phospho-Histone H2A.X (Ser139), eFluor 660, clone: CR55T33, eBioscience™
Mouse Monoclonal Antibody
Manufacturer: Life Technologies LS50986541
Histones are relatively small proteins that contain a high proportion of positively charged amino acids, e.g., arginine and lysine. This positive charge allows histones to bind tightly to the negatively charged DNA. There are five basic types of histone molecules called H1, H2A, H2B, H3, and H4. Histones are abundantly present in virtually all eukaryotic cells.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 15kDa 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/Transcirption Factor Buffer Set (cat. 00-5523) 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 Best Protocols 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 fluorochome.Excitation: 633-647 nm; Emission: 668 nm; Laser: Red Laser.Filtration: 0.2μm post-manufacturing filtered.
|Phospho-Histone H2A.X (Ser139)|
|PBS with 0.1% gelatin, 0.2% BSA and 0.09% sodium azide; pH 7.2|
|Histone H2A type 2-C, H2A 2C, H2AFQ, HIST2H2AC, Histone H2A/q|
|Flow Cytometry, Immunocytochemistry, Immunofluorescence|
|IgG1, kappa, kappa|
|Store at 2-8°C. Do not freeze. Light-sensitive material.|
For Research Use Only.