The SNAP-ChIP K-MetStat Panel (EpiCypher Cat. No. 19-1001) was used to analyze the performance of H3K4me3 recombinant polyclonal antibody (Cat. No. 711958) in ChIP. SNAP-ChIP panels consist of a pool of DNA-barcoded recombinant nucleosomes harboring unique histone post-translational modifications (PTMs, on- and off-target) that are spiked into a ChIP reaction early in the workflow. The K-MetStat panel includes unmethylated, mono-, di-, and tri-methyl forms of H3K4, H3K9, H3K27, H3K36, and H4K20 nucleosomes. Recovery of each unique DNA-barcoded nucleosome is quantified to determine how much of each PTM is immunoprecipitated in the ChIP reaction. H3K4me3 antibody was tested in native ChIP with 3 µg K-652 cell chromatin and 3 µg antibody. Specificity (left Y-axis) was determined by quantitative real-time PCR (qPCR) to each modified nucleosome in the SNAP-ChIP K-MetStat panel (X-axis). The black bar represents antibody efficiency (right Y-axis; log scale) and indicates percentage of the barcoded nucleosome target immunoprecipitated relative to Input. All bars represent mean ± SEM.
Histones are proteins that package genomic DNA into nucleosomes. This compaction allows for around two meters of DNA to fit into a cell’s nucleus. Nucleosomes contain two subunits, each made of histones H2A, H2B, H3, or H4, forming a histone octamer. Histones are subject to many forms of post-translational modifications (PTMs) which serve as epigenetic marks to signal activation or repression of gene expression and to alter chromatin compaction. These modifications lead to steric changes in chromatin structure that regulate various cellular processes such as transcription, replication, and DNA repair. Our growing portfolio of traditional and recombinant antibodies is designed to enable detection and characterization of histone modifications.
PTMs relevant to epigenetics research
- 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. In addition to acetylation, there are other forms of acylation such as crotonylation and butyrylation, the roles of which are just being investigated.
- 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 marker.
Products for western blot and ChIP applications
All the products below have been validated* by either peptide array or SNAP-ChIP (Sample Normalization and Antibody Profiling Chromatin Immunoprecipitation) and tested to work in the applications listed–see the application section 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 (WB). SNAP-ChIP, a spike-in of recombinant nucleosomes into a ChIP (Chromatin Immunoprecipitation) experiment, assays for epitopes that recognize the modification in the context of a nucleosome and is very good at qualifying antibodies for applications where epitopes are in native conformations, like ChIP. Learn more about our antibody validation procedures
More histone antibodies are continuously being developed and qualified, so check back if you don’t see an antibody of interest listed for a specific target in these tables.
Modification | SKU | Recommended for WB | Recommended for ChIP |
---|---|---|---|
H3K4me1 | 710795 | ✓ | ✓ |
H3K4me1 | 703946 | ✓ | ✓ |
H3K4me2 | 701764 | ✓ | ✓ |
H3K4me2 | 710796 | ✓ | ✓ |
H3K4me3 | 703954 | ✓ | ✓ |
H3K4me3 | 711958 | ✓ | ✓ |
H3K9me1 | MA5-33385 | ✓ | |
H3K9me1 | 710814 | ✓ | |
H3K9me2 | 710815 | ✓ | |
H3K9me3 | 49-1008 | ✓ | |
H3K9me3 | 713008 | ✓ | ✓ |
H3K27me1 | 49-1012 | ✓ | |
H3K27me1 | 712817 | ✓ | ✓ |
H3K27me1 | 712855 | ✓ | |
H3K27me1 | 703825 | ✓ | ✓ |
H3K27me2 | PA5-96115 | ✓ | |
H3K27me3 | MA5-11198 | ✓ | |
H3K27me3 | 713010 | ✓ | ✓ |
H3K36me1 | 701766 | ✓ | |
H3K36me2 | MA5-14867 | ✓ | ✓ |
H3K36me3 | MA5-24687 | ✓ | ✓ |
H3R2me1 | 703943 | ✓ | ✓ |
H3R2me2s | 712893 | ✓ | |
H3R8me2a | 712895 | ✓ | |
H3R17me2a | 712898 | ✓ | |
H4K20me1 | MA5-18067 | ✓ | |
H4K20me1 | PA5-17027 | ✓ | |
H4K20me1 | 712989 | ✓ | ✓ |
H4K20me2 | 720085 | ✓ | |
H4K20me3 | 701777 | ✓ | |
H4K20me3 | 703863 | ✓ |
Modification | SKU | Recommended for WB | Recommended for ChIP |
---|---|---|---|
H3K4ac | MA5-24673 | ✓ | ✓ |
H3K4ac | 703913 | ✓ | ✓ |
H3K4ac | 712905 | ✓ | ✓ |
H3K5ac | 703914 | ✓ | |
H3K9ac | MA5-33384 | ✓ | |
H3K9ac | 701269 | ✓ | |
H3K9ac | 703893 | ✓ | ✓ |
H3K9cr | MA5-33080 | ✓ | |
H3K14ac | MA5-24668 | ✓ | |
H3K14ac | 703894 | ✓ | ✓ |
H3K14ac | 712886 | ✓ | ✓ |
H3K18ac | 703896 | ✓ | ✓ |
H3K18ac | 712888 | ✓ | ✓ |
H3K18ac | 720095 | ✓ | |
H3K18cr | 703472 | ✓ | |
H3K23ac | MA5-24670 | ✓ | |
H3K27ac | MA5-23516 | ✓ | |
H3K27ac | 720096 | ✓ | |
H3K27cr | 712478 | ✓ | ✓ |
H3K36ac | MA5-24672 | ✓ | ✓ |
H4K5ac | MA5-32009 | ✓ | |
H4K5ac | PA5-40085 | ✓ | |
H4K5ac | 712906 | ✓ | |
H4K8ac | 701796 | ✓ | ✓ |
H4K8ac | 710828 | ✓ | ✓ |
H4K12ac | MA5-33388 | ✓ | |
H4K12ac | 701797 | ✓ | |
H4K12ac | 712991 | ✓ | ✓ |
H4K16ac | MA5-27794 | ✓ | |
H4K16ac | 720083 | ✓ | |
H4K20ac | MA5-33387 | ✓ | |
H4K20ac | 710810 | ✓ |
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.
Chromatin Immunoprecipitation (ChIP) assay of endogenous H3K4me3 protein was performed using H3K4me3 Recombinant Rabbit polyclonal Antibody (Cat. No. 711958, 5 µg) on sheared chromatin from 2 x 106 HeLa cells using the MAGnify ChIP System kit (Cat. No. 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to GAPDH gene and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.
Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot of Phospho-Histone H3 (Ser10) using Phospho-Histone H3 (Ser10) Antibody (9HCLC) Recombinant Rabbit Polyclonal (Cat. No. 710282) shows enrichment of Phospho-Histone H3 (Ser10) in HeLa cells upon Calyculin A treatment.
Antibody specificity for modified targets can be established using peptide arrays by quantifying the detection of the target histone modification and evaluating cross-reactivity with other histone modifications. Using Phospho-Histone H3 (Ser10) Recombinant Rabbit Polyclonal Antibody (Cat. No. 710282), an array of the specific peptide and other relevant peptides demonstrates that the antibody specifically recognizes the H3pS10 modification with minimal cross-reactivity to other histone modification peptides.
Resources
Behind the Bench: PRMTs: Role in epigenetic regulation
Epigenetics Antibody Poster
Antibody Learning Center: Epigenetics
Chromatin Immunoprecipitation (ChIP)—5 steps for great results
Chromatin Immunoprecipitation webinar
Bioprobes: SNAP-ChIP: A robust method for determining histone antibody specificity in ChIP
Poster: SNAP-ChIP A novel platform for ChIP standardization and antibody development
Overview of Western Blotting
* The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.