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29-668: Briefly, the conventional method is as follows: There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly cross-linked chromatin sheared by sonication called cross-linked ChIP (XChIP). Native ChIP (NChIP) uses native chromatin sheared by micrococcal nuclease digestion. Cross-linked ChIP is mainly suited for mapping the DNA target of transcription factors or other chromatin-associated proteins, and uses reversibly cross-linked chromatin as starting material. The agent for reversible cross-linking could be formaldehyde or UV light . Then

58-423: A permanent wave to hair involves the breaking and reformation of disulfide bonds. Typically a mercaptan such as ammonium thioglycolate is used for the breaking. Following this, the hair is curled and then "neutralized". The neutralizer is typically an acidic solution of hydrogen peroxide, which causes new disulfide bonds to form, thus permanently fixing the hair into its new configuration. Compromised collagen in

87-677: A much wider range of properties than conventional cross-linked elastomers because the domains that act as cross-links are reversible, so can be reformed by heat. The stabilizing domains may be non-crystalline (as in styrene-butadiene block copolymers) or crystalline as in thermoplastic copolyesters. Alkyd enamels , the dominant type of commercial oil-based paint, cure by oxidative crosslinking after exposure to air. In contrast to chemical cross-links, physical cross-links are formed by weaker interactions. For example, sodium alginate gels upon exposure to calcium ions, which form ionic bonds that bridge between alginate chains. Polyvinyl alcohol gels upon

116-459: A polymer depend strongly on the cross-link density. Low cross-link densities increase the viscosities of polymer melts . Intermediate cross-link densities transform gummy polymers into materials that have elastomeric properties and potentially high strengths. Very high cross-link densities can cause materials to become very rigid or glassy, such as phenol-formaldehyde materials. In one implementation, unpolymerized or partially polymerized resin

145-461: A technique developed for large-scale, de novo analysis of higher-order chromatin structures. Cross-link In chemistry and biology , a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers (such as proteins ). In polymer chemistry "cross-linking" usually refers to

174-434: A very efficient method for determining these factors, but there is a rivaling method known as ChIP-on-chip. ChIP-on-chip , also known as ChIP-chip, is an experimental technique used to isolate and identify genomic sites occupied by specific DNA-binding proteins in living cells. ChIP-on-chip is a relatively newer technique, as it was introduced in 2001 by Peggy Farnham and Michael Zhang. ChIP-on-chip gets its name by combining

203-471: Is a highly crosslinked polymer that comprises the main structural material of higher plants. A hydrophobic material, it is derived from precursor monolignols . Heterogeneity arises from the diversity and degree of crosslinking between these lignols. Intrastrand DNA crosslinks have strong effects on organisms because these lesions interfere with transcription and replication . These effects can be put to good use (addressing cancer) or they can be lethal to

232-680: Is a major disadvantage, which has led to the more predominant use of ChIP-chip in laboratories across the world. Table 1 Advantages and disadvantages of NChIP and XChIP Better chromatin and protein revery efficiency due to better antibody specificity In 1984 John T. Lis and David Gilmour, at the time a graduate student in the Lis lab, used UV irradiation, a zero-length protein-nucleic acid crosslinking agent, to covalently cross-link proteins bound to DNA in living bacterial cells. Following lysis of cross-linked cells and immunoprecipitation of bacterial RNA polymerase, DNA associated with enriched RNA polymerase

261-503: Is an experimental technique used to identify transcription factor binding events throughout an entire genome . Knowing how the proteins in the human body interact with DNA to regulate gene expression is a key component of our knowledge of human diseases and biological processes. ChIP-seq is the primary technique to complete this task, as it has proven to be extremely effective in resolving how proteins and transcription factors influence phenotypical mechanisms. Overall ChIP-seq has risen to be

290-550: Is commonly used to prepare antibody-hapten conjugates for antibody development. An in-vitro cross-linking method is PICUP ( photo-induced cross-linking of unmodified proteins ). Typical reagents are ammonium persulfate (APS), an electron acceptor, the photosensitizer tris-bipyridylruthenium (II) cation ( [Ru(bpy) 3 ] ). In in-vivo crosslinking of protein complexes, cells are grown with photoreactive diazirine analogs to leucine and methionine , which are incorporated into proteins. Upon exposure to ultraviolet light,

319-426: Is established by the specific site of the protein binding identification. The main difference comes from the efficacy of the two techniques, ChIP-seq produces results with higher sensitivity and spatial resolution because of the wide range of genomic coverage. Even though ChIP-seq has proven to be more efficient than ChIP-chip, ChIP-seq is not always the first choice for scientists. The cost and accessibility of ChIP-seq

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348-491: Is mainly suited for mapping the DNA target of histone modifiers. Generally, native chromatin is used as starting chromatin. As histones wrap around DNA to form nucleosomes, they are naturally linked. Then the chromatin is sheared by micrococcal nuclease digestion, which cuts DNA at the length of the linker, leaving nucleosomes intact and providing DNA fragments of one nucleosome (200bp) to five nucleosomes (1000bp) in length. Thereafter, methods similar to XChIP are used for clearing

377-462: Is reversed and proteins are removed by digestion with proteinase K . An epitope -tagged version of the protein of interest, or in vivo biotinylation can be used instead of antibodies to the native protein of interest. The DNA associated with the complex is then purified and identified by polymerase chain reaction (PCR), microarrays ( ChIP-on-chip ), molecular cloning and sequencing, or direct high-throughput sequencing ( ChIP-Seq ). Native ChIP

406-780: Is treated with a crosslinking reagent . In vulcanization , sulfur is the cross-linking agent. Its introduction changes rubber to a more rigid, durable material associated with car and bike tires . This process is often called sulfur curing. In most cases, cross-linking is irreversible, and the resulting thermosetting material will degrade or burn if heated, without melting. Chemical covalent cross-links are stable mechanically and thermally. Therefore, cross-linked products like car tires cannot be recycled easily. A class of polymers known as thermoplastic elastomers rely on physical cross-links in their microstructure to achieve stability, and are widely used in non-tire applications, such as snowmobile tracks, and catheters for medical use. They offer

435-641: The cross-links are likely to involve lysine e-amino groups in the N-terminals, disrupting the epitopes. This is likely to explain the consistently low efficiency of XChIP protocols compared to NChIP. But XChIP and NChIP have different aims and advantages relative to each other. XChIP is for mapping target sites of transcription factors and other chromatin-associated proteins; NChIP is for mapping target sites of histone modifiers (see Table 1). Chromatin Immunoprecipitation sequencing, also known as ChIP-seq ,

464-606: The native state . Common crosslinkers include the imidoester crosslinker dimethyl suberimidate, the N-Hydroxysuccinimide -ester crosslinker BS3 and formaldehyde . Each of these crosslinkers induces nucleophilic attack of the amino group of lysine and subsequent covalent bonding via the crosslinker. The zero-length carbodiimide crosslinker EDC functions by converting carboxyls into amine-reactive isourea intermediates that bind to lysine residues or other available primary amines. SMCC or its water-soluble analog, Sulfo-SMCC,

493-399: The addition of borax through hydrogen bonding between boric acid and the polymer's alcohol groups. Other examples of materials which form physically cross-linked gels include gelatin , collagen , agarose , and agar agar . Crosslinking is often measured by swelling tests. The crosslinked sample is placed into a good solvent at a specific temperature, and either the change in mass or

522-500: The cell debris, immunoprecipitating the protein of interest, removing protein from the immunoprecipitated complex, and purifying and analyzing the complex-associated DNA. The major advantage of NChIP is antibody specificity. Most antibodies to modified histones are raised against unfixed, synthetic peptide antigens. The epitopes they need to recognize in the XChIP may be disrupted or destroyed by formaldehyde cross-linking , particularly as

551-520: The change in volume is measured. The more crosslinking, the less swelling is attainable. Based on the degree of swelling, the Flory Interaction Parameter (which relates the solvent interaction with the sample), and the density of the solvent, the theoretical degree of crosslinking can be calculated according to Flory's Network Theory. Two ASTM standards are commonly used to describe the degree of crosslinking in thermoplastics. In ASTM D2765,

580-552: The cornea, a condition known as keratoconus , can be treated with clinical crosslinking. In biological context crosslinking could play a role in atherosclerosis through advanced glycation end-products (AGEs), which have been implicated to induce crosslinking of collagen, which may lead to vascular stiffening. Proteins can also be cross-linked artificially using small-molecule crosslinkers. This approach has been used to elucidate protein–protein interactions . Crosslinkers bind only surface residues in relatively close proximity in

609-505: The cross-linked chromatin is usually sheared by sonication, providing fragments of 300 - 1000 base pairs (bp) in length. Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin. Chromatin fragments of 400 - 500bp have proven to be suitable for ChIP assays as they cover two to three nucleosomes . Cell debris in the sheared lysate is then cleared by sedimentation and protein–DNA complexes are selectively immunoprecipitated using specific antibodies to

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638-414: The crosslinking agents vary greatly. Crosslinking generally involves covalent bonds that join two polymer chains. The term curing refers to the crosslinking of thermosetting resins, such as unsaturated polyester and epoxy resin, and the term vulcanization is characteristically used for rubbers . When polymer chains are crosslinked, the material becomes more rigid. The mechanical properties of

667-833: The distribution of histone H4 on heat shock genes using formaldehyde cross-linking. This technique was extensively developed and refined thereafter. NChIP approach was first described by Hebbes et al ., 1988, and has also been developed and refined quickly. The typical ChIP assay usually takes 4–5 days and requires 10~ 10 cells at least. Now new techniques on ChIP could be achieved as few as 100~1000 cells and completed within one day. ChIP has also been applied for genome-wide analysis by combining with microarray technology ( ChIP-on-chip ) or second-generation DNA-sequencing technology ( Chip-Sequencing ). ChIP can also combine with paired-end tags sequencing in Chromatin Interaction Analysis using Paired End Tag sequencing (ChIA-PET),

696-451: The host organism. The drug cisplatin functions by formation of intrastrand crosslinks in DNA. Other crosslinking agents include mustard gas , mitomycin , and psoralen . In proteins , crosslinks are important in generating mechanically stable structures such as hair and wool , skin , and cartilage . Disulfide bonds are common crosslinks. Isopeptide bond formation is another type of protein crosslink. The process of applying

725-412: The methods of Chromatin Immunoprecipitation and DNA microarray , thus creating ChIP-on-chip. The two methods seek similar results, as they both strive to find protein binding sites that can help identify elements in the human genome. Those elements in the human genome are important for the advancement of knowledge in human diseases and biological processes. The difference between ChIP-seq and ChIP-chip

754-413: The protein(s) of interest. The antibodies are commonly coupled to agarose , sepharose , or magnetic beads. Alternatively, chromatin-antibody complexes can be selectively retained and eluted by inert polymer discs. The immunoprecipitated complexes (i.e., the bead–antibody–protein–target DNA sequence complex) are then collected and washed to remove non-specifically bound chromatin, the protein–DNA cross-link

783-417: The sample is weighed, then placed in a solvent for 24 hours, weighed again while swollen, then dried and weighed a final time. The degree of swelling and the soluble portion can be calculated. In another ASTM standard, F2214, the sample is placed in an instrument that measures the height change in the sample, allowing the user to measure the volume change. The crosslink density can then be calculated. Lignin

812-433: The use of cross-links to promote a change in the polymers' physical properties. When "crosslinking" is used in the biological field, it refers to the use of a probe to link proteins together to check for protein–protein interactions , as well as other creative cross-linking methodologies. Although the term is used to refer to the "linking of polymer chains" for both sciences, the extent of crosslinking and specificities of

841-488: Was hybridized to probes corresponding to different regions of known genes to determine the in vivo distribution and density of RNA polymerase at these genes. A year later they used the same methodology to study the distribution of eukaryotic RNA polymerase II on fruit fly heat shock genes. These reports are considered the pioneering studies in the field of chromatin immunoprecipitation. XChIP was further modified and developed by Alexander Varshavsky and co-workers, who examined

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