Confocal immunofluorescent analysis of HeLa cells using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Alexa Fluor® 555 Conjugate) (red). Actin filaments were labeled with Alexa Fluor® 488 Phalloidin #8878 (green).
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This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 182
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (Alexa Fluor® 555 Conjugate) 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.
Human, Mouse, Rat, Monkey, D. melanogaster, S. cerevisiae
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.
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).
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