Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAbProduct information
Product Pathways - Chromatin Regulation / Epigenetics
Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725
|9725S||100 µl (10 western blots)||---||In Stock||---|
|9725T||20 µl (2 western blots)||---||In Stock||---|
|9725||carrier free and custom formulation / quantity||email request|
|W||1:1000||Human, Mouse, Rat, Monkey||Endogenous||17||Rabbit IgG|
Species cross-reactivity is determined by western blot.
Applications Key: W=Western Blotting, IP=Immunoprecipitation, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry), ChIP=Chromatin IP, ChIP-seq=Chromatin IP-seq
Specificity / Sensitivity
Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb detects endogenous levels of histone H3 when di-methylated on Lys4. This antibody shows weak cross-reactivity with histone H3 that is mono-methylated on Lys4 but does not cross-react with non-methylated or tri-methylated histone H3 Lys4. In addition, the antibody does not cross-react with methylated histone H3 Lys9, Lys27, Lys36 or 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 di-methylated.
Western blot analysis of whole cell lysates from HeLa, NIH/3T3, C6 and COS cells using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human stomach carcinoma using Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb.
Confocal immunofluorescent analysis of HeLa cells using Di-Methyl-Histone H3 (Lys4) (C64G9) 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 10 μl of Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across GAPDH, a known target gene of H3K4me2 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of Di-Methyl-Histone H3 (Lys4) (C64G9) 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.
Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb specificity was determined by peptide ELISA. The graph depicts 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 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, R546-51.
- Kubicek, S. et al. (2006) Ernst Schering Res Found Workshop , 1-27.
- Lin, W. and Dent, S.Y. (2006) Curr Opin Genet Dev 16, 137-42.
- Lee, D.Y. et al. (2005) Endocr Rev 26, 147-70.
- Daniel, J.A. et al. (2005) Cell Cycle 4, 919-26.
- Shi, X. et al. (2006) Nature 442, 96-9.
- Wysocka, J. et al. (2006) Nature 442, 86-90.
- Wysocka, J. et al. (2005) Cell 121, 859-72.
- Trojer, P. and Reinberg, D. (2006) Cell 125, 213-7.
- Peng, D. et al. (2011) PLoS One 6, e22009. Applications: Chromatin IP.
- Ohhata, T. et al. (2011) Genes Dev 25, 1702-15. Applications: Chromatin IP.
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7727 Biotinylated Protein Ladder Detection Pack
- 1434 Di-Methyl-Histone H3 (Lys4) Blocking Peptide
- 9717 Histone H3 (3H1) Rabbit mAb
- 4499 Histone H3 (D1H2) XP® Rabbit mAb
- 4620 Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated)
- 9715 Histone H3 Antibody
- 2650 Histone H3 Antibody (ChIP Formulated)
- 9723 Mono-Methyl-Histone H3 (Lys4) Antibody
- 5425 Normal Goat Serum
- 9701 Phospho-Histone H3 (Ser10) Antibody
- 6883 SignalFire™ ECL Reagent
- 12757 SignalFire™ Elite ECL Reagent
- 12630 SignalFire™ Plus ECL Reagent
- 8112 SignalStain® Antibody Diluent
- 9002 SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads)
- 9003 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads)
- 9727 Tri-Methyl-Histone H3 (Lys4) Antibody
- 9997 Tris Buffered Saline with Tween® 20 (TBST-10X)
U.S. Patent No. 5,675,063.
Ultra is a trademark of New England Biolabs, Inc.
Illumina is a registered trademark of Illumina, Inc.
NEBNext is a registered trademark of New England Biolabs, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.