The antibody was purified by affinity chromatography, and conjugated with Alexa Fluor® 488 under optimal conditions. The solution is free of unconjugated Alexa Fluor® 488.
Storage & Handling:
The antibody solution should be stored undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For immunofluorescent staining, the suggested use of this reagent is 5 µl per million cells. It is recommended that the reagent be titrated for optimal performance for each application.
* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm. ** Alexa Fluor® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.
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)
Nocodazole-treated Hela cells intracellularly stained with 2F3 Alexa Flour® 488
Description:
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.
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, C. H., et al., 2002. Proc. Natl. Acad. Sci. USA 99:8173. 4. Reina-San-Martin, B., et al., 2003. J. Exp. Med. 197:1767.
*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/ordering/). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.
