Tri-Methyl-Histone H4 (Lys20)

5737:

ChIP Agarose , ChIP Magnetic , Western Blotting

Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb recognizes endogenous levels of histone H4 protein only when tri-methylated at Lys20. This antibody does not cross-react with non-methylated, mono-methylated or di-methylated histone H4 Lys20. This antibody detects a 95 kDa non-specific protein of unkown origin.

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H4 in which Lys20 is tri-methylated.

Western Blotting

Western blot analysis of extracts from HeLa and NIH/3T3 cells using Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAb.

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 10⁶ HeLa cells and either 10 μl of Tri-Methyl-Histone H4 (Lys20) (D84D2) 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 α Satellite Repeat Primers #4486, SimpleChIP^® Human AFM Intron 1 Primers #5098, and SimpleChIP^® Human GAPDH Exon 1 Primers #5516. 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.

ELISA-Peptide

Tri-Methyl Histone H4 (Lys20) (D84D2) Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated tri-methyl histone H4 (Lys20) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the tri-methyl histone H4 (Lys20) peptide 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).

1. Peterson, C.L. and Laniel, M.A. (2004) Curr. Biol. 14, R546-R551.
2. Kubicek, S. et al. (2006) Ernst Schering Res. Found Workshop , 1-27.
3. Lin, W. and Dent, S.Y. (2006) Curr. Opin. Genet. Dev. 16, 137-142.
4. Lee, D.Y. et al. (2005) Endocr. Rev. 26, 147-170.
5. Daniel, J.A. et al. (2005) Cell Cycle 4, 919-926.
6. Shi, X. et al. (2006) Nature 442, 96-99.
7. Wysocka, J. et al. (2006) Nature 442, 86-90.
8. Wysocka, J. et al. (2005) Cell 121, 859-872.
9. Trojer, P. and Reinberg, D. (2006) Cell 125, 213-217.

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• 7003 20X LumiGLO^® Reagent and 20X Peroxide
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For Research Use Only. Not For Use In Diagnostic Procedures.