Cross-linked ChIP

This approach, also known as ChIP-on-chip, has been the most used technique to establish histone-maps. Briefly, as we have been detailing, the chromatin fragments are incubated in the presence of an antibody that recognizes a specific histone modification (e.g., methylation on histone 3 or acetylation on histone 4, etc.) (Figure 4). Next, the protein-DNA complex is immunoprecipitated. After reversal of the cross-link, ChIP-enriched DNA and control DNA are amplified by PCR and labeled with fluorescent dyes (Cy3 and Cy5). Finally, the samples are hybridized onto a specific microarray. The ratio of the Cy5 to Cy3 intensities measured for each DNA sequence in the array is a measure of the amount of a specific histone bound to the DNA. 

Histone modifications and how they are affected by toxicants can be investigated by chromatin immunoprecipitation (ChIP). Generally, there are two basic ChIP procedures (a) native ChIP (nChIP) that is performed on native digested chromatin and (b) cross-linked ChIP (xChIP), that includes a reversible chemical cross-link step to stabilize weaker DNA protein binding. The latter can be problematic as the epitope of the antibody may be blocked or destroyed by the cross-link. However, xChIP is more convenient, as samples can be collected and frozen and then precipitated all at the same time, whereas for nChIP the samples need to be precipitated directly. This is a major issue especially when analyzing time courses of PTM events. 

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

Often a cross-linking step is applied to stabilize the interactions between proteins and DNA. Thus, ChIP can be divided into (i) native ChIP ( N-ChIP ) without cross-linking and (ii) X-ChIP with cross-linking. Here we discuss briefly these two ways of performing ChIP. 

Herr, A.E., Singh, A.K., Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing. Anal. Chem. 2004, 76, 4727-4733. 

A primer surfacer, composed most often of a polyester to be cross-linked with a blocked isocyanate or melamine cross-linker, is applied next at a dry film thickness of approximately 1.0 mil by conventional spray application. The function of this coating is to provide filling and leveling properties as well as some stone-chip resistance. Cure conditions range from 250 to 325°F for 20 min. 

The first enzymatically coupled FET is sensitive to penicillin (1). It is composed of two separate FET chips and a silver/silver chloride reference electrode (see Fig. 4). Penicillinase, which produces protons during catalysis, is immobilized on one of the FET chips by utilizing the glutaraldehyde protein cross-linking reaction. The membrane of the reference FET chip contains immobilized bovine serum albumin. 

Microcrystalline cellulose may also contain low levels of non-saccharide organic residues. These emanate from lignin, a cross-linked bioploymer made up primarily of the three allylic alcohols/phenols in the wood chip starting material (Fig. 

Affinity Enrichment ChIP-based methods have been mostly combined with microarrays. These affinity-based techniques are now rapidly shifting to analysis by the next-generation techniques (56,66). As it has been described earlier, DNA immunoprecipitated with the specific methylcytosine antibody is de-cross-linked and defragmented. After ligation with specific probes, the sample is applied for on-site deep sequencing. 

Biological replication and appropriate controls are essential to ChIP experiments to assess reproducibility because of the nonrandom shearability of DNA and off-target effects of enrichment. A minimum of two replicates are recommended for both experimental and control samples. Control samples consist of two types a library consisting of formaldehyde-treated and -sheared DNA without antibody (input control) and a library where nonspecific, nonnuclear antibody is used for enrichment to the cross-linked DNA (mock library). Peaks in mapped reads from the enrichment library can be compared to peaks found from mapped reads control libraries to separate real and falsepositive peaks (Figure 4A). 

Applications of TMA are in many ways the simplest of the thermal techniques. Only the change in the size or position of a sample is being measured. However, they are also incredibly important in supplying information needed to design and process everything from chips to food products to engines. A sample of ASTM methods for TMA is shown in Table 1. Because of the sensitivity of modern TMA, it is often used to measure Tg s that are difficult to obtain by DSC, for example, those of highly cross-linked thermosets. 

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