Life Science Industry
The vast field of research known as "life science" looks at every living thing on the planet. Life sciences seek to understand all aspect of life on Earth, from microorganisms to begonias to beluga whales.
The term "life sciences industry" refers to companies, associations, and academic institutions that work to preserve and advance organismal life. A few of the businesses that fall under the umbrella of life sciences include biomedical research, pharmaceuticals, biophysics, neuroscience, cell biology, biotechnology, nutraceuticals, food processing, cosmeceuticals, and environmental sciences etc.
What is Chromatin Immunoprecipitation (ChIP)?
ChIP, an antibody-based technique, is used to selectively enrich particular DNA-binding proteins and the DNA that those proteins are intended to bind. ChIP is used to investigate specific protein-DNA interactions, multiple protein-DNA interactions, interactions over the entire genome, or interactions across a subset of genes.
ChIP uses antibodies that specifically detect and bind proteins, including as histones, histone modifications, transcription factors, and cofactors, to reveal information about chromatin states and gene transcription. Understanding gene expression and regulation in target cells or tissues is possible using molecular biology methods combined with proteomic analysis in ChIP.
How does ChIP works?
ChIP can be used to measure changes in interaction at particular stages of the cell cycle, after a treatment of interest, or to check for protein-DNA interaction at steady state. In live cells or tissues, protein and associated chromatin are momentarily cross-linked and sheared via enzymatic digestion or sonication to produce DNA fragments of between 300 and 1000 bp. Using a particular antibody, the protein of interest is immunoprecipitated from the cell debris together with any associated DNA fragments. DNA fragments are then purified when the cross-link is reversed. Using primers that surround the genomic locus of interest, quantitative real-time PCR can be used to measure the amount of eluted DNA. DNA amplification is a sign that the protein of interest is more enriched when it binds.
Different types of Chromatin Immunoprecipitation
The beginning chromatin preparation differs between the two primary forms of ChIP. The first technique employs cross-linked ChIP, or sonicated chromatin that has been reversibly cross-linked (XChIP). Micrococcal nuclease-digested native chromatin is used in another type: native ChIP (NChIP).
Cross linked ChIP (XChIP): Reversibly cross-linked chromatin is used as the starting material for cross-linked ChIP, which is primarily used for mapping the DNA target of transcription factors or other chromatin-associated proteins. UV light or formaldehyde are two possible cross-linking agents that can be reversed. The cross-linked chromatin is next typically fragmented by sonication, yielding fragments that range in size from 300 to 1000 base pairs (bp). The chromatin has been sheared by mild formaldehyde crosslinking, followed by nuclease digestion. As they cover two to three nucleosomes, chromatin fragments of 400 to 500 bp have shown to be acceptable for ChIP experiments
Native linked ChIP (NChIP): Native ChIP is mostly useful for identifying the DNA sites that histone modifiers modify. Native chromatin is typically employed as the starting chromatin. Proteins are crosslinked to the chromatin using a fixing agent. Instead, a nuclease-digested cell nucleus is used to separate natural chromatin. N-ChIP has the benefit of greater antibody recognition and binding to its target antigens since antibodies are generated against unfixed antigens.
The large quantity of histone proteins may make PCR for downstream analysis unnecessary. N-ChIP is an approach that is appealing due to these benefits, but it can only be used to find histones. Additionally, the steps of chromatin breakdown and immunoprecipitation may result in loss of protein binding, which could skew the results or prevent accurate assessments.
Limitation of ChIP
ChIP has several limits of its own, as do any molecular biology methods. ChIP assays frequently produce weak signals when compared to controls, producing findings that cannot be concluded. It is challenging to pinpoint the precise binding site of a protein since the assay is constrained to a resolution related to the size of the DNA fragments created after shearing. While ChIP can infer the existence of a protein at a certain genomic locus, it cannot determine if the protein's binding to that DNA sequence has any functional importance. Additionally, proteins of no functional significance that momentarily interact with DNA or DNA-binding proteins may also form cross-links. Similar to this, it's possible to miss protein interactions with DNA that have a brief residence period (as low as a few seconds for some transcription factors). Additionally, the protein of interest's epitope may be concealed by interacting proteins.
Finally, the ChIP approach may not be able to distinguish between distinct DNA-binding protein isoforms since it is so dependent on the caliber and specificity of the antibody used.
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