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This simple guide will help you choose the techniques and reagents most suited to your experiments
all the way from sample preparation to modification identification and characterization.
Whether using cells or tissues, well-prepared starting material is essential for good data. The table below summarizes the different types of extraction, the application each one is best suited to, and the kits we have developed to help you achieve this.
|Whole cell extraction||Nuclear extraction||Nuclear extraction (nucleic acid-free)||Histone extraction||Chromatin extraction|
|Applications||Enzyme activity assay|
|Enzyme activity assay|
|Protein detection||Histone detection||Chromatin IP|
DNA-protein binding assays
Nuclear enzyme assays
|Sample type and amount||Cells: 2-5 million|
Cells: 2-5 million
Cells: 2-5 million
Cells: 2-5 million
Tissue: 10 mg
Cells: 0.1-10 million
Tissue: 50-200 mg
|Assay time||≤ 45 min||≤ 60 min||≤ 60 min||≤ 60 min||≤ 60 min|
The initial step in investigating histone post-translational modifications (PTMs) is often to look at total changes in the level of PTMs across the whole genome. Antibodies against specific proteins or protein modifications are available for use in western blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), and ELISA techniques.
For example, our histone WB protocol can be used to compare total histone PTMs in disease vs healthy samples. In this case, a nuclear control antibody like anti-histone H3 (ab1791), would be used to normalize the results.
Histone PTMs can be also quantified with specific assays with colorimetric or fluorometric readouts, providing a quick way of scaling up your experiments if you have large sample cohorts.
Chromatin Immunoprecipitation (ChIP) allows you to identify where histone modifications are in the genome. ChIP uses antibodies to isolate a protein or modification of interest, together with any bound DNA. This can then be used to identify where the protein or modification of interest is located within the genome and its relative abundance at each location.
ChIP’ing histone modifications is a powerful tool to analyze chromatin structure and gene expression. For example, H3K9me3 marks heterochromatin and satellite repeats, H3K27me3 promoters in gene-rich regions, H3K4me1 active enhancers, and RNA pol II phospho S2 and S5 correlate with initiation and elongation fo transcription respectively.
Our ChIP kits allow you to perform high quality, reproducible ChIP.
The most important factor in a ChIP experiment is the antibody.
|ChIP-grade antibodies to key histone modifications and related proteins|
|H4K16ac||RNA pol II phopsho S2||RNA pol II phopsho S5||RNA pol II|
|View all ChIP grade antibodies in our expanding range|
Addition and removal of histone modifications is carried out by enzymes called writers and erasers. Their activity can be determined using enzyme activity assays. Applications include the characterization of histone modification pathways both in the context of fundamental epigenetic mechanisms or drug discovery, where compounds (ie potential inhibitors) can be screened against a panel of assays.
For our specialized kits to quantify the enzyme activity of writers and erasers, see our guide to assays for histone methylation and demethylation.
The table below details some of the writer and eraser enzymes involved in specific histone PTMs.
|Modification||Human recombinant proteins|
|H3K4 demethylation||KDM1A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, PHF8, C14-orf169/NO66|
|H3K27 methylation||EZH1, EZH2, NSD2, NSD3, G9A, EHMT1|
|H3K27 demethylation||PHF8, KDM6A, KDM6B, KDM7A|
|Histone deacetylation||HDAC1 to 11|
inhibition of these regulatory enzymes using small molecules can be useful to probe the biological functions of histone modifications. To find out more about how these compounds work, see our guide to histone H3 methyltransferase and demethylase inhibitors. Some compounds inhibit the function of histone modification binding proteins (also known as ‘readers’). For example, JQ1 inhibits the interaction of bromodomains in the BET protein family with acetylated lysines.
Inhibitors of writers, erasers and readers key tools for understanding epigenetic modification pathways are also essential for the validation of ‘druggable’ targets in the context of pre-clinical studies both in academia and pharmaceutical industry.
View the full list of inhibitors here.
Mass spectrometry (MS) has become an essential tool to characterize histone PTMs1. It has the power to identify multiple novel modifications in single peptides by using a slightly modified version of the traditional MS bottom-up approach2. This involves chemical derivatization of samples to increase sequence coverage. PTMs induce a mass shift that is visible in the MS spectra: for example, +14 Da for methyl and +142 Da for acetyl groups.
High-resolution analyzers like Orbitraps are commonly used to carry out histone PTM analysis due to their power to distinguish between PTMs with nearly identical mass signatures (acetylation at 42.0106 Da and tri-methylation at 42.0470 Da for example)3.
Further MS/MS fragmentation and liquid chromatography elution experiments are used to validate the newly identified modifications in vivo, often side-by-side with heavy isotope labeling and antibody detection.
1. Karch, K. R., DeNizio, J. E., Black, B. E. & Garcia, B. A. Identification and interrogation of combinatorial histone modifications. Front. Genet. 4, 1–15 (2013).
2. Arnaudo, A. R., Garcia, B.A. Proteomic characterisation of novel post-translational histone modifications. Epigenetics and Chromatin 6:24 (2013).
3. Karch, K. R., Zee, B. M. & Garcia, B. A. High Resolution Is Not a Strict Requirement for Characterization and Quanti fi cation of Histone Post-Translational Modi fi cations. J. Proteome Res. (2014).