Antibodies for Epigenetics Research

Epigenetics is the study of heritable changes in gene expression that modify DNA, RNA, and protein – but do not alter the primary sequence. Post-translational modifications (PTMs) are the most common methods for regulating epigenetic states. Many types of proteins are subject to PTMs and one of the most highly decorated is histones. Our growing portfolio of traditional and recombinant antibodies is designed to enable detection and characterization of epigenetics targets with exceptional specificity to particular PTMs.

PTMs relevant to epigenetics research

Histones are proteins that package genomic DNA into nucleosomes. This allows for around 2 meters of DNA to fit into a cell’s nucleus. Nucleosomes contain 2 subunits each made of histones H2A, H2B, H3, or H4. Histones are highly subject to a variety of PTMs at specific amino acid residues on the histone tails that protrude from the core nucleosome. These modifications lead to steric changes in chromatin structure that regulate various cellular processes such as transcription, replication, and DNA repair.

  • Methylation—one, two, or three methyl groups are added to lysine or arginine. In the case of histones, histone methyltransferases (HMTs) have specificity for the histone residues and mono-, di-, or trimethylation.
  • Acetylation—the acetyl group from acetyl coenzyme A is added to specific histone lysine residues by histone acetyltransferases (HATs) and acetyl groups are removed by specific histone deacetylases (HDACs). Acetylation is generally associated with gene activation.
  • Phosphorylation—kinases phosphorylate specific serines, threonines, or tyrosines on histones and dephosphorylation is carried out by phosphatases. Phosphorylation often occurs during DNA repair and mitosis.
  • Ubiquitination—ubiquitin is added by an E3 ligase and removed by a deubiquitinating enzyme (DUB). Although, ubiquitination often is a mark for protein degradation, in this case ubiquitination is an epigenetic mark.

Products for western blot and ChIP applications

All of the products below have been validated by either peptide array or SNAP-ChIP and tested to work in the shown applications – see the application section below for data examples. Peptide array testing shows epitopes or linearized peptides and is very good at qualifying antibodies for applications where epitopes are denatured, like western blot. A SNAP-ChIP assay shows epitopes that are properly folded and is very good at qualifying antibodies for applications where epitopes are in native confirmations, like ChIP. Learn more about our antibody validation procedures

Applications

Our growing portfolio of traditional and recombinant antibodies is designed to enable detection and characterization of epigenetics targets with exceptional specificity to particular post-translational modifications and reproducibility in the form of antibody lot-to-lot consistency.

Western blot using epigenetic antibody for methyl-histone H3 (Lys4)
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Western blot analysis on acid extracts. Western blot was performed on 20 µg lysate of HeLa (Lane 1), K-562 (Lane 2), MDA-MB-231 (Lane 3), COS-7 (Lane 4) and A549 (Lane 5). The blot was probed with Anti-Methyl-Histone H3 (Lys4) Polyclonal Antibody (Cat. No. PA5-40087, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Cat. No. A27036, 0.25 µg/mL, 1:4000 dilution). A 17 kDa band corresponding to Methyl-Histone H3 (Lys4) was observed across the cell lines tested.

Chromatin Immunoprecipitation (ChIP) on sheared chromatin. ChIP was performed using Anti- Histone H3K4me2 Recombinant Rabbit Oligoclonal Antibody (Cat. No. 710796, 5 µg) on sheared chromatin from 2 million HeLa cells using the "MAGnify ChIP system" kit (Cat. No. 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Cat. No. 4351106) with optimized PCR primer pairs for the promoters of the active cFOS, beta-actin genes, and IRF1 Exon 2 region used as positive control target gene, and the region of the inactive SAT2 satellite repeat used as negative control target gene. Data is presented as fold enrichment of the Antibody signal versus the negative control IgG using the comparative CT method.

Chromatin Immunoprecipitation (ChIP) using epigenetic antibody for Histone H3K4me2
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Peptide array using recombinant polyclonal antibody
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Histone H3K4me1 detection with superior peptide specificity. Peptide arrays were performed using our recombinant polyclonal antibody (Cat. No. 710795) and two other suppliers’ antibodies targeting H3K4me1. Arrays were incubated overnight with a 1:2,000 dilution of primary antibody. After washing, they were incubated with goat anti–rabbit IgG (H+L) Superclonal secondary antibody (Cat. No. A27036) at a dilution of 1:5,000 for 1 hr. Arrays were incubated with SuperSignal West Pico PLUS substrate (Cat. No. 34578).

SNAP-ChIP K-MetStatTM (EpiCypher, 19-1001) was performed to analyze the histone specificity of H3K4me3 (Cat. No. 710795) in ChIP. The ChIP assay spikes in a panel of post-translationally modified, barcoded, semi-synthetic nucleosomes during the normal ChIP workflow The panel includes unmethylated, mono, di and tri-methyl forms of H3K4, H3K9, H3K27, H3K36 and H4K20 nucleosomes that can later be quantified to determine how much of each PTM is immunoprecipitated in the ChIP reaction. 710795 was tested in native ChIP with 3 µg chromatin from HEK-293 cells. Specificity (left Y-axis; all bars mean 177 SEM from six independent ChIP experiments) was determined by quantitative real-time PCR (qPCR) to each modified nucleosome in the SNAP-ChIP K-MetStat panel (X-axis). Black bar represents antibody efficiency (right Y-axis; log scale) and indicates percentage of the barcoded H3K4me1 nucleosome target immunoprecipitated relative to input.