Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IHC-P IF-IC ChIP H M R Mk (X) (Z) Endogenous 17 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  X=Xenopus  Z=Zebrafish
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb detects endogenous levels of histone H3 only when tri-methylated on Lys27. The antibody does not cross-react with non-methylated, mono-methylated or di-methylated Lys27. In addition, the antibody does not cross-react with mono-methylated, di-methylated or tri-methylated histone H3 at Lys4, Lys9, Lys36 or Histone H4 at Lys20.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys27 is tri-methylated.

Western Blotting

Western Blotting

Western blot analysis of various cell lines using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lymphoma using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb in the presence of control peptide (left) or antigen specific peptide (right).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 µl of Tri-Methyl-Histone H3 (Lys27) (C36B11) 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 MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human MYT-1 Exon 1 Primers #4493. 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

ELISA-Peptide

Tri-Methyl Histone H3 (Lys27) (C36B11) Rabbit mAb specificity was determined by peptide ELISA. The graph depicts the binding of the antibody to pre-coated tri-methyl histone H3 (Lys27) peptide in the presence of increasing concentrations of various competitor peptides. As shown, only the tri-methyl histone H3 (Lys27) peptide competed away binding of the antibody.

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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