Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Chromatin Regulation / Epigenetics

Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAb #4658

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC ChIP H M R Mk (Dm) (X) (Z) (B) (Pg) (Sc) (Ce) Endogenous 17 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Pg=Pig  Sc=S. cerevisiae  Ce=C. elegans
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Di-Methyl-Histone H3 (Lys9) (D85B4) XP™ Rabbit mAb detects endogenous levels of histone H3 only when di-methylated on Lys9. The antibody 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.

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 Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Di-Methyl-Histone H3 (Lys9) (D85B4) XP™ Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys9) (D85B4) XP™ Rabbit mAb(green). Actin filaments have been labeled with DY-554 phalloidin (red).

ELISA

ELISA

Di-Methyl Histone H3 (Lys9) (D85B4) 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 di-methyl histone H3 (Lys9) peptide competed away binding of the antibody.


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 20 μl of Di-Methyl-Histone H3 (Lys9) (D85B4) 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.

Background

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.

Application References

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