Streamlined chromatin studies workflows, to illustrate how an example study might progress and the EpiGentek kits to support these steps
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What is Chromatin Immunoprecipitation (ChIP)?
One of the most widely used methods for studying chromatin-related modifications is chromatin immunoprecipitation (ChIP), a process by which a protein of interest binds to a specific genomic DNA region. This popular ChIP assay provides epigenetic researchers with valuable information about the interaction among certain proteins or protein modifications and a specific DNA sequence.
Protein-DNA interactions are crucial to certain cellular functions such as DNA replication and recombination, gene transcription, chromosome segregation, signal transduction, and epigenetic silencing. Understanding protein-DNA interactions can be used, for example, to compare the amount of histone methylation linked to a specific gene promoter region among two different groups, such as tissues that are diseased or healthy. Chromatin immunoprecipitation methods can also be used to map the genomic location of various other histone modifications.
Is ChIP Right for Your Experiment?
Because chromatin immunoprecipitation and various downstream applications can take time and come with their associated expenses, it is worthwhile to find out if ChIP is a good fit for your study.
To begin, we recommend reviewing the existing literature that exists related to your proposed experiment. There are times when the legwork has been completed by other researchers giving your study a sturdy foundation to begin upon without having to reinvent the wheel. From here, variations and unique angles can be taken by your team to make the study interesting, unique, and worthwhile, while reviewing the previously completed work to discover any potential obstacles or problems the previous researchers faced. Let others’ experience work to your advantage to save time and headache from your own study in advance.
Lastly, there is a possibility that researchers have already done ChIP on your target, and therefore could save a considerable and invaluable amount of time and effort. A few of the most common resources where information like this can be found include USCS Genome Browser, the ENCODE Project, and the NCBI. Be sure to check these resources for information prior to conducting your study to see if the work has already been done. If it has not and your target has yet to be elucidated upon, undertaking a ChIP experiment could be the right solution for your study.
Chromatin is a combination of DNA and proteins that package DNA into a smaller volume to fit in the cell, helps to prevent DNA damage, and controls gene expression and DNA replication. Proper isolation of chromatin samples is an important factor in assays for studying protein-DNA interactions. EpiGentek's proprietary chromatin isolation kits ensure quality isolation of intact chromatin using a variety of methods to help extract chromatin from mammalian or plant cells and tissues.
For isolating chromatin or DNA-protein complex from mammalian cells or tissues in a simple and rapid format, followed by a shearing step by sonication
Chromatin Accessibility
Chromatin structure is an important determinant of gene regulation and one of the main factors involved is how accessible the chromatin is for transcription factors and other DNA binding proteins. Generally speaking, the more condensed the chromatin, the more difficult it is for transcription factors and other DNA binding proteins to access DNA and carry out their tasks. The more accessible the DNA, the more likely surrounding genes are actively transcribed. Researching the accessibility of chromatin’s structure is of particular interest to provide information into the presence or absence of nucleosomes, which directly or indirectly affects a range of other cellular and metabolic processes.
For quantitative chromatin analysis and determination of euchromatin or heterochromatin states
Chromatin Fragmentation
Ultrasonic energy processing of chromatin samples is a widely accepted alternative to enzymatic digestion methods. By emphasizing higher throughput processing and preventing sample contamination in our sonication technologies, our EpiSonic™ sonicators can be easily integrated into existing lab workflows and are also particularly suitable for compatibility with epigenetic and next-generation applications.
For multi-sample shearing of DNA and chromatin in next-generation sequencing applications
Chromatin Immunoprecipitation (ChIP)
Because protein-DNA interaction plays such a crucial role in cellular functions, identifying genetic targets of DNA binding proteins and knowing the mechanisms of protein-DNA interaction is critical for understanding cellular processes. One of the tools in researchers’ belts to studying these interactions is Chromatin Immunoprecipitation or ChIP. The advantage of ChIP over other methods is that this process allows for detecting a specific protein binding to specific sequences of a gene in living cells. EpiGentek provides a range of assay kit solutions to help your lab carry out successful ChIP-based experiments efficiently, accurately, and repeatedly.
Taking the process a step further, NGS (Next Generation Sequencing) technologies have increased data output volumes and lowered the cost of ChIP-sequencing. ChIP-Seq offers a combination of the chromatin immunoprecipitation process already discussed paired with next generation sequencing methods to offer an effective method for identifying genome-wide DNA binding sites of transcriptions factors and other proteins. ChIP-Seq offers the benefit of resolving the specific genetic sequence of target protein binding sites on a genome-wide level. Real-time quantitative PCR (RT-qPCR) offers an alternative approach by analyzing the binding sites of modified histones and/or other DNA-binding proteins at precise loci using selective amplification with specific primers. EpiGentek offers various inexpensive solutions for both PCR & NGS, including full package kits to conveniently construct high quality DNA libraries from low quantity or difficult samples.
For quantitative real time polymerase chain reaction (PCR) analysis of ChIP
CUT&RUN / CUT&TAG
In recent years, developments have been made that are revolutionizing how researchers perform DNA-protein interaction analysis through a process called CUT&RUN. Standing for Cleavage Under Target & Release Using Nuclease, the process was developed to release the captured target protein/DNA complex from limited biological materials for mapping protein-DNA interaction and has significantly improved mapping resolution. This process has allowed for less input materials to be used while limiting background signals. And as an extension, cTIP (CUT & Tag In Place), adds the step of preparing a library for next generation sequencing and provide a similar solution to ChIP-Seq with all the benefits previously mentioned. As the research landscape changes, CUT&RUN and cTIP are poised to become increasingly popular methods of DNA-protein interaction studies, given their wide range of benefits.
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