All tags Histone H3 Methods and tools to study histone modifications

Methods and tools to study histone modifications

Whether you are quantifying histone modifications, measuring or inhibiting the activity of writers and erasers, we can help you make your mark in epigenetic research.

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.

Getting started – sample preparation

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 extractionNuclear extractionNuclear extraction (nucleic acid-free)Histone extractionChromatin extraction
ApplicationsEnzyme activity assay

Protein detection
Enzyme activity assay

Protein detection
Protein detectionHistone detectionChromatin IP

DNA-protein binding assays

Nuclear enzyme assays
Sample type and amountCells: 2-5 million

Cells: 2-5 million

Tissue: variable

Cells: 2-5 million

Tissue: variable

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
Product codeab113475ab113474ab113477ab113476ab117152

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Quantifying global levels of histone modifications

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.

Related products


Detecting and quantifying modifications by ChIP

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
Histone H3H3K4me1H3K27me3H3K9me3
H3K27acH3K9acH3S10pH3R2cit
H3K27MH2AK119ubγH2A.XHistone H4
H4K16acRNA pol II phopsho S2RNA pol II phopsho S5RNA pol II
View all ChIP grade antibodies in our expanding range

ChIP resources


Measuring the activity of writers and erasers

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.

ModificationHuman recombinant proteins
H3K4 methylation

SETD7, NSD3, KMT2A, KMT2C, KMT2D, PRDM9, SETD1A, SETD1B, SMYD3 

H3K4 demethylationKDM1A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, PHF8, C14-orf169/NO66
H3K27 methylationEZH1, EZH2, NSD2, NSD3, G9A, EHMT1
H3K27 demethylationPHF8, KDM6A, KDM6B, KDM7A
Histone deacetylationHDAC1 to 11


Inhibiting writers, erasers and readers

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.

Related products:

  • JQ1, a potent, selective and cell-permeable BET bromodomain inhibitor
  • GSK-J4, a cell-permeable histone demethylase JMJD3/UTX inhibitor
  • MI-192 hydrochloride, a selective HDAC2/3 inhibitor

View the full list of inhibitors here.

Identifying novel histone modifications using mass spectrometry

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.​


References

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).


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