antibody

Histone H3, dimethyl (Lys9)

H5110-14D2

20 ul

monoclonal

domestic rabbit

nonconjugated

methylated

10E30(D85B4)

Histone H3, dimethyl (Lys9)

rabbit

Mab

Methylated

Antibodies

20 ul

10E30(D85B4)

IgG

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 recent discovery of histone demethylases such as PADI4, LSD1, JMJD1, JMJD2 and JHDM1 has shown that methylation is a reversible epigenetic mark (9). Applications: Suitable for use in Western Blot, Flow Cytometry, Immunofluorescence (IF-IC) and ChIP. Other applications not tested. Recommended Dilution: Western Blot: 1:1000. Incubate membrane with diluted antibody in 5% BSA, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight. Immunoprecipitation: 1:50 Immunofluorescence (IF-IC): 1:200 Chromatin Immunoprecipitation (ChIP): 1:50 Flow Cytometry: 1:100 Optimal dilutions to be determined by the researcher. Storage and Stability: May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

Hu Mk Mo Rt

Synthetic peptide corresponding to the N-terminus of histone H3 in which Lys9 is di-methylated. Species Sequence Homology: porcine, D. melanogaster, S. cerevisiae, bovine, xenopus, zebrafish, C. elegans.

Recognizes human Di-Methyl-Histone H3 (Lys9). Species crossreactivity: mouse, rat, monkey. Does not cross-react with non-methylated, mono-methylated or tri-methylated Histone H3 Lys9. In addition, the antibody does not cross-react with histone H3 di-methylated on Lys4, Lys27, or Lys36 and histone H4 di-methylated on Lys20.

Purified

Supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, 0.1mg/ml BSA, ≤0.02% sodium azide, 50% glycerol.

Not determined

Product Type: Mab Isotype: IgG Clone No: 10E30(D85B4) Host: rabbit Source: human Concentration: Not determined Form: Supplied as a liquid in 10mM sodium HEPES, pH 7.5, 150mM sodium chloride, 0.1mg/ml BSA, ≤0.02% sodium azide, 50% glycerol. Purity: Purified Immunogen: Synthetic peptide corresponding to the N-terminus of histone H3 in which Lys9 is di-methylated. Species Sequence Homology: porcine, D. melanogaster, S. cerevisiae, bovine, xenopus, zebrafish, C. elegans. Specificity: Recognizes human Di-Methyl-Histone H3 (Lys9). Species crossreactivity: mouse, rat, monkey. Does not cross-react with non-methylated, mono-methylated or tri-methylated Histone H3 Lys9. In addition, the antibody does not cross-react with histone H3 di-methylated on Lys4, Lys27, or Lys36 and histone H4 di-methylated on Lys20. Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.

ChIP FC IF WB

-20°C

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.

https://usbio-images.r.worldssl.net/prodimages/2/H5110-14D2_1.jpg

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4x10e6 HeLa cells and either 20ul of H5110-14D2 or 2ul of Normal Rabbit IgG using SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads). The enriched DNA was quantified by Real-Time PCR using primers specific for the transcriptionally active GAPDH and RPL30 genes, the inactive AFM gene, and the heterochromatic alpha Satellite repeat element. 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.

https://usbio-images.r.worldssl.net/prodimages/2/H5110-14D2_5.jpg

Flow cytometric analysis of HeLa cells using Di-Methyl-Histone H3 (Lys9) (H5110-14D2) (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP Isotype Control (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) was used as a secondary antibody.