Analysis Note

ControlHeLa acid extract

Application

Immunocytochemistry Analysis:
1:500 dilution from a representative lot detected Histone H3 in NIH/3T3 and A431 cells.

Dot Blot Analysis:
1:1,000 dilution from a representative lot specifically detected Histone H3 in a Lys4 dimethhyl modified histone H3 peptide while not detecting potential cross-reacting peptides corresponding to other modified and non-modified histones.

Chromatin Immunoprecipitation Analysis:
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 4 µg of either Normal IgG (Part No. 12-370), or 4 µL of Anti-Dimethyl Histone H3 (Lys4) (Part No. ABE250) and the Magna ChIP™ A/G Kit (Cat. # 17-10085). Successful immunoprecipitation of Dimethyl-Histone H3 (Lys4) associated DNA fragments was verified by qPCR using primers specific for the human GAPDH coding region.
Please refer to the EZ-Magna ChIP™ A/G (Cat. # 17-10085) protocol for experimental details.

ChIP-Sequencing
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (cat# 17-10460), 5 µg ofa representative lot of anti-dimethyl-Histone H3 (Lys4) antibody(ABE250), 20 µL Protein A/G beads ,and 5e6 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 twelve 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.

Research Sub CategoryHistones

Anti-dimethyl Histone H3 (Lys4) Antibody is a Rabbit Polyclonal Antibody for detection of Histone H3 dimethylated at lysine 4. This purified polyclonal antibody, also known as H3K4me2, has dot blot (DB) proven specificity & has been validated in WB, ICC, ChIP.

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

Histone H3 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H3 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. Dimethylation of histone H3 at Lys4 correlates with transcriptional activity of many genes.

Immunogen

Epitope: Dimethylated Lys4

KLH-conjugated linear peptide corresponding Histone H3 methylated at Lys4.

Linkage

Replaces: 04-791

Physical form

Affinity purified

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

Quality

Evaluated by Western Blot using HeLa acid extract and recombinant histone proteins.

Western Blot Analysis: 1:1,000 dilution of this antibody detected Histone H3 on 10 µg of HeLa acid extract.

Specificity

This antibody recognizes Histone H3 dimethylated at Lys4.

Broad species cross-reactivity expected based on sequence similarity.

Storage and Stability

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

Target description

~17 kDa observed