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Western blot analysis of H2AX (pS139) in transformed human epithelioid carcinoma (Left Panel). Lysates from control (left image) and Staurosporine (EMD Biosciences, Cat. No. 569397)-treated (right image) HeLa cells (ATCC CCL-2) were probed with Purified Mouse anti-H2AX (pS139) (Cat. No. 560443) at 0.25, 0.125, and 0.06 µg/ml (Lanes 1, 2, and 3, respectively). H2AX (pS139 )is identified as a band of ~15 kDa. Immunofluorescent staining of human cell line. HeLa cells (ATCC CCL-2) were seeded in a 96-well plate at ~10,000 cells per well (Middle Panels). After overnight culture, the cells were exposed to 2400 Joules UV irradiation (right image) or untreated (left image) and then allowed to recover for 30-60 minutes at 37°C. The cells were fixed, permeabilized with cold methanol, and stained with Purified Mouse anti-H2AX (pS139) followed by Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen, Cat. No. A-11029, pseudo colored green) according to the Recommended Assay Procedure. Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The images were captured on a BD Pathway™ 435 high-content Bioimager system using a 20X objective and merged using BD AttoVision™ software. This antibody also worked with the Saponin and the Triton™ X-100 Perm/Wash protocols (see Recommended Assay Procedure; Bioimaging protocol link). Flow cytometric analysis of H2AX (pS139) expression on human peripheral blood mononuclear cells (Right Panel). PBMCs were incubated at 37°C for 2 hours while untreated (dashed line histogram) or treated (solid line histogram) with 50 µm of Etoposide. Cells were fixed with Fixation Buffer (Cat. No. 554655) and permeabilized with Perm Buffer III (Cat. No. 558050). Cells were then stained with Purified Mouse anti-H2AX (pS139), followed by PE Goat Anti-Mouse Ig (Multiple Adsorption) (Cat. No. 550589). Fluorescence histograms were derived from gated events with the side and forward light-scattering characteristics of viable PBMCs.
Western blot analysis of H2AX (pS139) in transformed human epithelioid carcinoma (Left Panel). Lysates from control (left image) and Staurosporine (EMD Biosciences, Cat. No. 569397)-treated (right image) HeLa cells (ATCC CCL-2) were probed with Purified Mouse anti-H2AX (pS139) (Cat. No. 560443) at 0.25, 0.125, and 0.06 µg/ml (Lanes 1, 2, and 3, respectively). H2AX (pS139 )is identified as a band of ~15 kDa. Immunofluorescent staining of human cell line. HeLa cells (ATCC CCL-2) were seeded in a 96-well plate at ~10,000 cells per well (Middle Panels). After overnight culture, the cells were exposed to 2400 Joules UV irradiation (right image) or untreated (left image) and then allowed to recover for 30-60 minutes at 37°C. The cells were fixed, permeabilized with cold methanol, and stained with Purified Mouse anti-H2AX (pS139) followed by Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen, Cat. No. A-11029, pseudo colored green) according to the Recommended Assay Procedure. Cell nuclei were counterstained with Hoechst 33342 (pseudo colored blue). The images were captured on a BD Pathway™ 435 high-content Bioimager system using a 20X objective and merged using BD AttoVision™ software. This antibody also worked with the Saponin and the Triton™ X-100 Perm/Wash protocols (see Recommended Assay Procedure; Bioimaging protocol link). Flow cytometric analysis of H2AX (pS139) expression on human peripheral blood mononuclear cells (Right Panel). PBMCs were incubated at 37°C for 2 hours while untreated (dashed line histogram) or treated (solid line histogram) with 50 µm of Etoposide. Cells were fixed with Fixation Buffer (Cat. No. 554655) and permeabilized with Perm Buffer III (Cat. No. 558050). Cells were then stained with Purified Mouse anti-H2AX (pS139), followed by PE Goat Anti-Mouse Ig (Multiple Adsorption) (Cat. No. 550589). Fluorescence histograms were derived from gated events with the side and forward light-scattering characteristics of viable PBMCs.
BD Pharmingen™ Purified Mouse anti-H2AX (pS139)
BD Pharmingen™ Purified Mouse anti-H2AX (pS139)
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Bioimaging:
For further technical support, please refer to "Cellular Imaging" at our website: http://www.bdbiosciences.com/us/s/resources
1. Seed the cells in appropriate culture medium at an appropriate cell density in a Falcon™ 96-well Imaging Plate, and
culture overnight to 48 hours.
2. Remove the culture medium from the wells, and wash (one to two times) with 100 μl of 1× PBS.
3. Fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).
4. Remove the fixative from the wells, and wash the wells (one to two times) with 100 μl of 1× PBS.
5. Permeabilize the cells using either cold methanol (a), Triton™ X-100 (b), or Saponin (c):
a. Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
b. Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
c. Add 100 µl of 1× Perm/Wash buffer (Cat. No. 554723) to each well and incubate for 15 to 30 minutes at RT. Continue to use 1× Perm/Wash buffer for all subsequent wash and dilutions steps.
6. Remove the permeabilization buffer from the wells, and wash one to two times with 100 μl of appropriate buffer (either 1× PBS or 1× Perm/Wash buffer, see step 5.c.).
7. Optional blocking step: Remove the wash buffers, and block the cells by adding 100 µl of blocking buffer BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 3% FBS in appropriate dilution buffer to each well and incubating for 15 to 30 minutes at RT.
8. Dilute the antibody to its optimal working concentration in appropriate dilution buffer. Titrate purified (unconjugated) antibodies and second-step reagents to determine the optimal concentration. If using a Bioimaging Certified antibody conjugate, dilute it 1:10.
9. Add 50 µl of diluted antibody per well and incubate for 60 minutes at RT. Incubate in the dark if using fluorescently labeled antibodies.
10. Remove the antibody, and wash the wells three times with 100 μl of wash buffer. An optional detergent wash (100 μl of 0.05% Tween in 1× PBS) can be included prior to the regular wash steps.
11. If the antibody being used is fluorescently labeled, then move to step 12. Otherwise, if using a purified unlabeled antibody, repeat steps 8 to 10 with a fluorescently labeled second-step reagent to detect the purified antibody.
12. After the final wash, counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
13. View and analyze the cells on an appropriate imaging instrument.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Triton is a trademark of the Dow Chemical Company.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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.
Development References (4)
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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
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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
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Kuo LJ, Yang LX. Gamma-H2AX - A novel biomarker for DNA double-strand breaks. In Vivo. 2008; 22(3):305-309. (Biology). View Reference
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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
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