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Each lot of this H2A.X antibody is quality control tested by Western blotting. Western blotting, suggested working dilution(s): Use 5 μg antibody per 5 ml antibody dilution buffer for each mini-gel. For immunofluorescence microscopy: Use a dilution range of 1~4 µg/ml. It is recommended that the reagent be titrated for optimal performance for each application.
COA:
Application Notes:
Intracellular staining protocol for Anti-H2A.X-Phosphorylated (Ser139) Antibodyfor Flow Cytometry
1. Prepare 70% absolute ethanol. Chill solution by storing at -20C. 2. Prepare cells of interest. 3. Wash 1X: resuspend with PBS, then pellet cells by centrifugation (250Xg, 5min) 4. Discard the supernatant and vortex to loosen cell pellet. 5. Add pre-cooled 70% ethanol drop by drop, while vortexing. 6. Incubate at -20C for 60 minutes. 7. Wash 3X with BioLegend Cell Staining Buffer and resuspend the cells at 0.5-1 X 10^7/ml in the cell staining buffer. 8. Perform immunofluorescent staining.
Additional reported applications (for the relevant formats of this clone) include: immunohistochemistry on paraffin embedded sections2, immunofluorescence microscopy3-9, western blot 10-12, and flow cytometry1,13.
Application References:
1. Moiseenko V, et al. 2008 Radiat Oncol 3:18 (FC) 2. Akbay A,et al. 2008. Am J Pathol. 173:536. (IHC) PubMed 3. Mochizuki K, et al.2008.J cell Sci.121:2148. (IF) PubMed 4. Xiao R, et al. 2007. Mol Cell Biol.27:5393. (IF) PubMed 5. Rossi DJ, et al. 2007. Nature. 447:725. (IF) PubMed 6. Loidl J, et al. 2009. Mol Cell Biol. 20:2048. (IF) PubMed 7. Beels L, et al. 2009. Circulation. 120:1903. (IF) PubMed 8. Suzuki K, et al. 2010. Nucleic Acids Res. 38:e129. (IF) PubMed. 9. Lukaszewicz A. 2010. Chromasoma Apr 27. [Epub ahead of print] (IF) PubMed 10. Yamada C, et al. 2010 J. Biol. Chem. 285:16693. (WB) PubMed 11. Bu Y, et al. 2010, Biochem Biophys Res Commun. 397:157. (WB) PubMed 12. Massignan T, et al. 2010. J. Biol Chem. 285:7752. (WB) PubMed. 13. Banath JP, et al. 2010. BMC Cancer 10:4 (FC) 14. Suzuki K, et al. 2010. Nucleic Acids Res. 38:129. PubMed 15. Lee J, et al. 2011. J Cell Biol. 192:263. PubMed 16. Li M, et al. 2011. Mol Cell Biol. 31:2090. PubMed. 17. Martinelli P, et al. 2011. Blood. 117:6617. PubMed.
Untreated Hela cells (Panel A), or overnight nocodazole treated Hela cells (Panel B) stained with purified mouse monoclonal antibody against Ser139 phosphorylated H2A.X, followed by Rhodamine Red-X conjugated Donkey anti-mouse IgG and DAPI.
Untreated (Lane 1) and staurosporine-treated (Lane 2) Jurkat nuclear extract, untreated (Lane 3) and UV treated (Lane 4) Jurkat cell extract were western blotted using anti-phospho-H2A.X (Ser 139) antibody, clone: 2F3.
H2A.X is a 14 kD basal histone and a member of the H2 histone family. This nuclear protein is synthesized in the G1 and S phase of the cell cycle and is known to be important for recombination between immunoglobulin switch regions. H2A.X becomes phosphorylated on serine 139 after double-stranded DNA breaks. Phosphorylated H2A.X promotes DNA repair and maintains genomic stability. The 2F3 monoclonal antibody reacts with phosphorylated human H2A.X (Ser139) and has been shown to be useful for Western blotting and immunofluorescence staining.
Other Names:
H2A.x, H2a/x, Histone 2A, Histone 2A.X
Structure:
Basal histone, H2 histone family; 14 kD
Distribution:
Nuclear
Function:
Phosphorylated H2AX promotes DNA repair and maintains genomic stability. Important for recombination between immunoglobulin switch regions
Regulation:
Synthesized in G1 and S-phase of cell cycle
Modification:
Phosphorylation on Ser139 after double-stranded DNA breaks
Antigen References:
1. Mannironi C, et al.1989. Nucleic Acids Res. 17:9113. 2. Celeste A, et al. 2002. Science 296:922. 3. Bassing CH, et al. 2002. Proc. Natl. Acad. Sci. USA 99:8173. 4. Reina-San-Martin B, et al. 2003. J. Exp. Med. 197:1767.
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This data display is provided for general comparisons between formats. Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors. If you need assistance with selecting the best format contact our expert technical support team.
Purified anti-H2A.X-Phosphorylated (Ser139)
Untreated Hela cells (Panel A), or overnight nocodazole treated Hela cells (Panel B) stained with purified mouse monoclonal antibody against Ser139 phosphorylated H2A.X, followed by Rhodamine Red-X conjugated Donkey anti-mouse IgG and DAPI.
Untreated (Lane 1) and staurosporine-treated (Lane 2) Jurkat nuclear extract, untreated (Lane 3) and UV treated (Lane 4) Jurkat cell extract were western blotted using anti-phospho-H2A.X (Ser 139) antibody, clone: 2F3.
FITC anti-H2A.X-Phosphorylated (Ser139)
Staurosporine-treated (37oC, 4 hours) (filled green histogram) or non-treated Molt-4 cells (opened purple histogram) stained with 2F3 FITC
