Di-Methyl-Histone H3 (Lys4) An

9726:

ChIP Agarose , ChIP Magnetic , IHC / Paraffin , Immunofluorescence , Immunoprecipitation , Western Blotting

Di-Methyl-Histone H3 (Lys4) Antibody detects endogenous levels of histone H3 when di-methylated on Lys4. The antibody shows very slight cross-reactivity with mono-methylated Lys4, but does not cross-react with non-methylated or tri-methylated Lys4. In addition, the antibody does not cross-react with mono-methylated, di-methylated or tri-methylated histone H3 at Lys9, Lys27, Lys36 or histone H4 at Lys20.

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys4 is di-methylated. Antibodies are purified by peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from various cell lines using Di-Methyl-Histone H3 (Lys4) Antibody.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Di-Methyl-Histone H3 (Lys4) Antibody in the presence of control peptide (left) or antigen specific peptide (right).

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys4) Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4x10⁶ HeLa cells and either 20 μl of Di-Methyl-Histone H3 (Lys4) Antibody 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 RPL30 Exon 3 Primers #7014, SimpleChIP^® Human GAPDH Exon 1 Primers #5516, SimpleChIP^® Human MyoD1 Exon 1 Primers #4490, 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.

ELISA-Peptide

Di-Methyl-Histone H3 (Lys4) Antibody specificity was determined by peptide ELISA. The graphs depict the binding of the antibody to pre-coated di-methyl-histone H3 (Lys4) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the di-methyl-histone H3 (Lys4) peptide competed away binding of the antibody. The antibody does show slight cross-reactivity with mono-methyl-histone H3 (Lys4).

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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For Research Use Only. Not For Use In Diagnostic Procedures.