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Analysis Note

ControlHeLa nuclear extracts

Application

ChIP Analysis:
An independent lab has shown that this antibody performs in chromatin immunoprecipitation (ChIP).
Dot Blot Analysis: A 1:10,000 dilution from a representative lot detected trimethyl-Histone H3 (Lys 4 ) in an Absurance Histone H3 Antibody Specificity Array (Cat. No. 16-667) and an Absurance Histone H2A, H2B, H4 Antibody Specificity Array (Cat. No. 16-665).
ChIP-seq Analysis:
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (cat# 17-10460), 1 µL Anti-trimethyl-Histone H3 (Lys4) antibody (cat# 07-473) , 20 µL Protein A/G beads, and 1e6 crosslinked HeLa cell chromatin followed by DNA purification using magnetic beads. Libraries were prepared from Input and ChIP DNA samples using standard protocols with Illumina barcoded adapters, and analyzed on Illumina HiSeq instrument. An excess of eighteen million reads from FastQ files were mapped using Bowtie (http://bowtie-bio.sourceforge.net/manual.shtml) following TagDust (http://genome.gsc.riken.jp/osc/english/dataresource/) tag removal. Peaks were identified using MACS (http://luelab.dfci.harvard.edu/MACS/), with peaks and reads visualized as a custom track in UCSC Genome Browser (http://genome.ucsc.edu) from BigWig and BED files. The highest 25% of peaks identified in the 07-473 dataset showed 99% overlap with peaks identified in the ENCODE H3K4me3 BROAD Histone track for HeLa S3.

Research Sub CategoryHistones

Anti-trimethyl-Histone H3 (Lys4) Antibody is a rabbit polyclonal antibody for detection of Histone H3 trimethylated at lysine 4. Also known as Anti-H3K4me3, this highly specific and well published antibody has been validated in ChIP, DB, WB, PIA, ChIP-seq.

Research CategoryEpigenetics & Nuclear Function

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. Histone modifications regulate DNA transcription, repair, recombination, and replication. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which DNA is wrapped in repeating units, called nucleosomes, which limits DNA accessibility to the cellular machineries, which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. Histone H3 contains a main globular domain and a long N-terminal tail and is involved with the structure of the nucleosomes of the ′beads on a string′ structure. The N-terminal tail of histone H3 protrudes from the globular nucleosome core and can undergo several different types of epigenetic modifications that influence cellular processes. These modifications include the covalent attachment of methyl or acetyl groups to lysine and arginine amino acids and the phosphorylation of serine or threonine. H3K4me3 modifications are reported to occur consistently at transcription start sites and H3K4me3 domain is associated with higher transcription activity and cell identity in pre-implantation development and in the process of deriving embryonic stem cells from the inner cell mass and trophoblast stem cells from the trophectoderm. (Ref.: Liu, X., et al. (2016). Nature 537(7621); 558-562).

Immunogen

Epitope: Trimethylated (Lys4)

Synthetic peptide corresponding to residues surrounding and including trimethlyated Lys 4 of Histone H3.

Legal Information

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Linkage

Replaces: 04-791

Physical form

Affinity purified

Affinity purified rabbit polyclonal antibody in buffer containing 0.1M Tris-Glycine (pH 7.4), 150mM NaCl with 0.05% sodium azide.

Quality

Evaluated by Western Blotting on HeLa acid extracted nuclear preps (positive) and recombinant Histone H3 (negative).

Western Blotting Analysis:
1:5,000 – 1:10,000 dilution of this antibody was used to detect trimethylated-Histone H3 (Lys4) in HeLa nuclear acid extracts.

Specificity

Expected to react with most other species based on extreme conserved homology among species.

This antibody only detects Histone H3 when trimethylated on Lysine 4.

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

Target description

~17 kDa observed; 15.51 kDa calculated. Uncharacterized bands may be observed in some lysate(s).