Product Pathways - Chromatin Regulation / Epigenetics
Pan-Methyl-Histone H3 (Lys9) (D54) XP® Rabbit mAb #4473
|4473S||100 µl (10 western blots)||---||In Stock||---|
|4473P||40 µl (4 western blots)||---||In Stock||---|
|4473||carrier free and custom formulation / quantity||email request|
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|W||1:1000||Human, Mouse, Rat, Monkey||Endogenous||17||Rabbit IgG|
Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, ChIP=Chromatin IP
Species predicted to react based on 100% sequence homology: Chicken, D. melanogaster, Xenopus, Zebrafish, Bovine, Pig, S. cerevisiae, C. elegans.
* Product-specific protocol.
Specificity / Sensitivity
Pan-Methyl-Histone H3 (Lys9) (D54) XP™ Rabbit mAb detects endogenous levels of histone H3 only when mono-, di-, or tri-methylated on Lys9. The antibody does not cross-react with histone H3 methylated on Lys4, 27 and 36, or histone H4 methylated on Lys20.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys9 is di-methylated.
Western blot analysis of extracts from various cell lines using Pan-Methyl-Histone H3 (Lys9) (D54) XP™ Rabbit mAb.
Flow cytometric analysis of human whole blood cells using Pan-Methyl-Histone H3 (Lys9) (D54) XP® Rabbit mAb (blue) and Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody. Analysis was performed on cells in the lymphocyte gate.
Confocal immunofluorescent analysis of HeLa cells using Pan-Methyl-Histone H3 (Lys9) (D54) XP® Rabbit mAb (green). Actin filaments were labeled using DY-554 phalloidin (red).
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 20 μl of Pan-Methyl-Histone H3 (Lys9) (D54) XP® Rabbit mAb or 2 μl of Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human AFM Intron 1 Primers #5098, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Pan-Methyl-Histone H3 (Lys9) (D54) XP™ Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated di-methyl histone H3 (Lys9) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the mono-, di- and tri-methyl histone H3 (Lys9) peptides competed away binding of the antibody.
The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1). Histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development (2,3). Arginine methylation of histones H3 (Arg2, 17, 26) and H4 (Arg3) promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases (PRMTs), including the co-activators PRMT1 and CARM1 (PRMT4) (4). In contrast, a more diverse set of histone lysine methyltransferases has been identified, all but one of which contain a conserved catalytic SET domain originally identified in the Drosophila Su(var)3-9, Enhancer of zeste, and Trithorax proteins. Lysine methylation occurs primarily on histones H3 (Lys4, 9, 27, 36, 79) and H4 (Lys20) and has been implicated in both transcriptional activation and silencing (4). Methylation of these lysine residues coordinates the recruitment of chromatin modifying enzymes containing methyl-lysine binding modules such as chromodomains (HP1, PRC1), PHD fingers (BPTF, ING2), tudor domains (53BP1), and WD-40 domains (WDR5) (5-8). The discovery of histone demethylases such as PADI4, LSD1, JMJD1, JMJD2, and JHDM1 has shown that methylation is a reversible epigenetic marker (9).
- Peterson, C.L. and Laniel, M.A. (2004) Curr. Biol. 14, R546-R551.
- Kubicek, S. et al. (2006) Ernst Schering Res. Found Workshop, 1-27.
- Lin, W. and Dent, S.Y. (2006) Curr. Opin. Genet. Dev. 16, 137-142.
- Lee, D.Y. et al. (2005) Endocr. Rev. 26, 147-170.
- Daniel, J.A. et al. (2005) Cell Cycle 4, 919-926.
- Shi, X. et al. (2006) Nature 442, 96-99.
- Wysocka, J. et al. (2006) Nature 442, 86-90.
- Wysocka, J. et al. (2005) Cell 121, 859-872.
- Trojer, P. and Reinberg, D. (2006) Cell 125, 213-217.
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For Research Use Only. Not For Use In Diagnostic Procedures.
XP® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.