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PE Mouse Anti-H2AX (pS139)
PE Mouse Anti-H2AX (pS139)
Flow cytometric analysis of H2AX (pS139) expression in human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were either not treated (solid line histogram) or cultured with 50 μM Etoposide (Calbiochem, Cat. No. 341205) for 2 hrs at 37°C (dashed line histogram). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No.558050). After washing with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), the cells were stained with PE Mouse Anti-H2AX (pS139) antibody (Cat. No. 562377). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of H2AX (pS139) expression in human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were either not treated (solid line histogram) or cultured with 50 μM Etoposide (Calbiochem, Cat. No. 341205) for 2 hrs at 37°C (dashed line histogram). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No.558050). After washing with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), the cells were stained with PE Mouse Anti-H2AX (pS139) antibody (Cat. No. 562377). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Product Details
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BD Pharmingen™
H2A.X; H2A/X; H2AFX; HIST5-2AX; gamma-H2AX; γ-H2AX; H2AX (pS140)
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG1, κ
Phosphorylated Human H2AX Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2737611
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
562377 Rev. 2
Antibody Details
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N1-431

Histones are highly basic proteins that complex with DNA to form chromatin. The H2AX histone (~15 kDa calculated molecular weight) is a member of the H2A histone family whose members are components of nucleosomal histone octamers. Double-stranded breaks in DNA caused by replication errors, apoptosis, or other physiological processes (including, immunoglobulin and TCR gene recombinations) and DNA damage caused by ionizing radiation, UV light, or cytotoxic agents lead to phosphorylation of H2AX on serine 139. H2AX (pS139) is also referred to as H2AX (pS140) when the N-terminal methionine that is normally excised during posttranslational processing is included in amino acid sequence numbering. Kinases such as ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) phosphorylate H2AX to induce its function. Phosphorylated H2AX (also termed, gamma-H2AX) functions to recruit and localize DNA repair proteins or cell cycle checkpoint factors to the DNA-damaged sites. In this way, phosphorylated H2AX promotes DNA repair and maintains genomic stability and thus helps prevent oncogenic transformations. Immunofluorescent staining and bioimaging analysis of cultured cells can be used to readily identify H2AX (pS139)-containing foci. As such, H2AX (pS139) immunofluorescence localization serves as a biomarker for nuclear sites of DNA damage (e.g., double-stranded DNA breaks) in affected cells.

562377 Rev. 2
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
562377 Rev.2
Citations & References
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Development References (5)

  1. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem. 2001; 276(45):42462-42467. (Biology). View Reference
  2. Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A. H2AX: the histone guardian of the genome. DNA Repair (Amst). 2004; 3(8-9):959-967. (Biology). View Reference
  3. Kuo LJ, Yang LX. Gamma-H2AX - A novel biomarker for DNA double-strand breaks. In Vivo. 2008; 22(3):305-309. (Biology). View Reference
  4. Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem. 2000; 275(13):9390-9395. (Biology). View Reference
  5. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998; 273(10):5858-5868. (Biology). View Reference
View All (5) View Less
562377 Rev. 2

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.