Product Pathways - Chromatin Regulation / Epigenetics
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751
|9751S||100 µl (10 western blots)||---||In Stock||---|
|9751||carrier free and custom formulation / quantity||email request|
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|W||1:1000||Human, Mouse, Rat, Monkey, D. melanogaster, S. cerevisiae||Endogenous||17||Rabbit IgG|
Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, ChIP=Chromatin IP
Species predicted to react based on 100% sequence homology: Xenopus, Zebrafish.
* Product-specific protocol.
Specificity / Sensitivity
Tri-Methyl-Histone H3 (Lys4) Antibody detects endogenous levels of histone H3 when tri-methylated on Lys4. This antibody shows some cross-reactivity with histone H3 that is di-methylated on Lys4, but does not cross-react with non-methylated or mono-methylated histone H3 Lys4. In addition, the antibody does not cross-react with methylated histone H3 Lys9, Lys27, Lys36 or methylated histone H4 Lys20.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of histone H3 in which Lys4 is tri-methylated.
Antibody specificity was determined by Western blotting. HeLa and NIH/3T3 cell lysates were probed with Tri-Methyl Histone H3 (Lys4) (C42D8) Rabbit mAb (Panel A) or Tri-Methyl Histone H3 (Lys4) Rabbit mAb pre-adsorbed with 1.5 μM of various competitor peptides (Panels B-M). As shown, only the tri-methyl histone H3 (Lys4) peptide competed away binding of the antibody.
Western blot analysis of various cell types using Tri-Methyl Histone H3 (Lys4) (C42D8) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon using Tri-Methyl-Histone H3 (K4) (C42D8) Rabbit mAb in the presence of non-methyl peptide (left) or K4 tri-methyl peptide (right).
Flow cytometric analysis of human whole blood cells using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (blue) and Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody. Analysis was performed on cells in the lymphocyte gate.
Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of Tri-Methyl-Histone H3 (Lys4) (C42D8) 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 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.
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).
- Peterson, C.L. and Laniel, M.A. (2004) Curr. Biol. 14, R546-R551.
- Kubicek, S. et al. (2006) Ernst Schering Res. Found Workshop, 1-27.
- Lin, W. and Dent, S.Y. (2006) Curr. Opin. Genet. Dev. 16, 137-142.
- Lee, D.Y. et al. (2005) Endocr. Rev. 26, 147-170.
- Daniel, J.A. et al. (2005) Cell Cycle 4, 919-926.
- Shi, X. et al. (2006) Nature 442, 96-99.
- Wysocka, J. et al. (2006) Nature 442, 86-90.
- Wysocka, J. et al. (2005) Cell 121, 859-872.
- Trojer, P. and Reinberg, D. (2006) Cell 125, 213-217.
- Pasini, D. et al. (2010) Nucleic Acids Res 38, 4958-69. Applications: Western Blotting.
- Schmitz, S.U. et al. (2011) EMBO J 30, 4586-600. Applications: Western Blotting, Chromatin IP.
- Peach, S.E. et al. (2012) Mol Cell Proteomics 11, 128-37. Applications: Chromatin IP.
- Thurman, R.E. et al. (2012) Nature 489, 75-82. Applications: Chromatin IP.
- Paige, S.L. et al. (2012) Cell 151, 221-32. Applications: Chromatin IP.
- Pasini, D. et al. (2010) Nature 464, 306-10. Applications: Chromatin IP.
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For Research Use Only. Not For Use In Diagnostic Procedures.
Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
This antibody is developed, validated, and produced by CST using in part technology under license (granting certain rights including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.