Anti-Dimethyl Histone H3 (Lys4) Antibody, clone CMA303

Histones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome. Histones are modified post-translationally; and these modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark, and dimethyl histone H3 Lys4 (H3K4me2) is depleted from regions with DNA methylation. Multipotential hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3-). These genes are transcriptionally silent and highly enriched in lineage-specific hematopoietic genes, suggesting a role for H3K4 methylation in differentiation.

Broad species cross-reactivity is expected, based on sequence homology.

This antibody specifically recognizes Histone H3 dimethylated at Lys4. The antibody binding specificity allows for phosphorylation of Thr3 and/or modifications of Lys91.

Epitope: Dimethylated Lys4

Synthetic peptide corresponding to amino acids 1-12 of human Histone H3, dimethylated on Lys4, conjugated to KLH.

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 106 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either a normal mouse IgG, or Anti-dimethyl-Histone H3 (Lys4) antibody and the Magna ChIP G (Cat. # 17-611) Kit. Successful immunoprecipitation of dimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding.
Please refer to the EZ-Magna G ChIP (Cat. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.

Dot Blot Analysis:
Absurance Histone H3 Antibody Specificity Array (Cat. No. 16-667) and Absurance Histone H2A, H2B, H4 Antibody Specificity Array (Cat. No. 16-665), which contain histone peptides with various modifications were probed with Cat. No 05-1338, Anti-dimethyl H3 (Lys4), clone CMA303 at 2.0 µg/mL (1:500 dilution). Proteins were visualized using a Donkey anti-mouse IgG conjugated to HRP and a chemiluminescence detection system.

ChIP-seq Analysis:
Chromatin immunoprecipitation was performed using the Magna ChIP HiSens kit (cat# 17-10460), 2 µg of Anti-dimethyl-Histone H3 (Lys4) antibody (cat# 05-1338), 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 sixteen 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 04-790 and 05-1338 datasets showed 92 and 90% overlap with peaks identified in the ENCODE H3K4me2 BROAD Histone track for HeLa S3.

Immunocytochemistry:
This antibody has been shown by an outside laboratory to be suitable for immunocytochemistry.

Immunoprecipitation:
This antibody has been shown by an outside laboratory to be suitable for immunoprecipitation.

ELISA:
This antibody has been shown by an outside laboratory to be suitable for ELISA

Multiplexing:
This antibody specifically recognizes histone H3 dimethylated on Lys4 by Luminex assay.

Anti-Dimethyl Histone H3 (Lys4) Antibody, clone CMA303 is a mouse monoclonal antibody for detection of Dimethyl Histone H3 (Lys4) also known as H3K4me2, Histone H3 (di methyl K4). Demonstrated performance in ChIP, DB, ELISA, ICC, IP, WB, Mplex.

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Histones

Routinely evaluated by Western Blot on HeLa acid extract.

Western Blot Analysis: 2 μg/mL (1:500) dilution of this antibody detected dimethyl Histone H3 (Lys4) on 10 μg of HeLa acid extract.

approx. 17 kDa

Format: Purified

Protein G Purified

Purified mouse monoclonal IgG1κ in PBS with 0.05% sodium azide.

Stable for 1 year at 2-8°C from date of receipt. For maximum recovery of product, centrifuge the vial prior to removing the cap. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Control
HeLa Acid extract lysate

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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.