Developed by Epicypher and available in Europe through tebu-bio, CUTANA CUT&RUN and its sister technology CUT&TAG are quickly becoming the preferred approaches for Chromatin Mapping experiments over the classical method ChiP-Seq. To supplement these advances, EpiCypher is also leading the way for the validation of highly specific antibodies to histone PTMs for chromatin profiling assays. Let’s check how these new antibodies are validated.
So, what precisely were researchers looking for when asking to “run histone binding studies”?
1. The determination of the exact modification on a specific histone recognized by an antibody raised against histones. In fact, this could be meant as a strigent quality control experiment for an antibody to be used to recognize specific histone modifications. Furthermore, such a specific antibody could be used to block out the binding of any other protein to the histone target sequence. As the 4 histones which make the nucleosome’s histone octamer (H2A, H2B, H3, and H4) can be epigenetically modified by a number of chemical reactions, such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP Ribosylation, theoretically thousands of modification patterns can be found on histones.2. The determination of specific binding sites of epigenetic “readers”, such as bromodomains (recognizing acetylated sites) and methyl readers. These “readers” can be domains of “writers” (enzymes modifying histones, e.g. methyl transferases and acetylases) or “erasers” (e.g. demethylases and deacetylases). Thus, these modifying enzymes can be targeted to their sites of action on histones. 3. Furthermore, a third application field for peptide arrays containing diverse modified and non modified histone sequences could be the analysis of the substrate specificity of entire histone modifying enzymes. All these applications could help in answering questions in research fields and diseases which are linked to epigenetic histone modifications such as cancer, aging, asthma, diabetes, cardiovascular diseases, neurodegeneration, autoimmune disorders, and developmental disorders.
We found arrays to answer all these questions: Precise histone binding studies are now possible with EpiCypher’s EpiTitan Histone Peptide ArraysThe EpiTitan™ Histone Peptide Arrays represent the next generation in histone peptide arrays, and are the gold standard against which all other histone peptide arrays are judged. The arrays feature hundreds of highly purified and well-characterized histone peptides spotted onto the substrate. EpiTitan™ Histone Peptide Arrays are a dramatic improvement over arrays in which the peptides are synthesized on the substrate (SPOT synthesis), a technique that leads to peptides on the array that are of unknown quantity and quality. All the peptides on the EpiTitan™ Histone Peptide Arrays are purified by HPLC and validated by mass spectrometry prior to spotting. The technique used in this array is quite easy.
- Biotinylated peptides are spotted 12 times each per array on a surface covered with streptavidin to catch the peptides.
- Each array slide comes with two subarrays, thus two samples per slide can be evaluated.
- The protein to be investigated is added and binds to its specific target sequence and modification pattern.
- The detection of binding either starts with an antibody against the protein of interest or an anti tag antibody, should the protein carry the respective tag, and can be visualized using fluorescence scanning or standard ECL techniques – by the way the EpiTitan™ Histone Peptide Array is the only array available on the market which supports both detection methods.
- Protocols for qualitative as well as quantitative analysis of the data are provided.