Nonspecific binding, to proteins

As indicated in Figure 3A, selenide, which can nonspecifically bind to proteins, is a central selenium species in the pathways leading to the formation and degradation of selenium proteins. Selenide is also formed from selenite by reduction via glutathione. This reaction occurs in rat (Gasiewicz and Smith 1978) and human (Lee et al. 1969) red blood cells, as well as in human plasma containing added glutathione (Mas and Sarker 1989). 

Figure 2.3 SAM surface modification has been done using monothiol and dithiol compounds containing PEG linkers. Useful coatings typically contain mainly PEG-hydroxyl or PEG-monomethyl ether linkers that provide a biocompatible lawn, which prevents nonspecific binding of proteins to the metallic surface. About 10 percent of the surface modifications are done using a longer carboxylate-containing thiol-PEG linker that provides sites for attachment of affinity ligands.

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

CNTs can be functionalized with protein via non-covalent bond (Li et al., 2005 Kim et al., 2003 Mitchell et al., 2002). For example, (beta-lactamase I, that can be immobilized inside or outside CNTs, doesn t change enzyme s activity (Vinuesa and Goodnow, 2002). Taq enzyme can attach to the outside of CNT, and doesn t change its activity (Cui et al., 2004). Peptide with Histidine and Tryptophan can have selective affinity for CNT(Guo et al., 1998). Monoclonal antibody can attach to SWNTs. Protein-modified CNTs can be used to improve its biocompatibility and biomolecular recognition capabilities (Um et al., 2006). For example, CNTs functionalized with PEG and Triton X-100 can prevent nonspecific binding of protein and CNTs. Biotin moiety is attached to the PEG chains Streptavidin can bind specifically with biotin-CNT (Shim et al., 2002). 

The dye Coomassie Brilliant Blue R250 nonspecifically binds to all the protein. The gel is soaked in the dye for it to seep in and bind to the proteins. The gel is then destained to remove the unbound dye. The dye binds to the protein and not the gel, and hence the protein bands can be visually seen. The binding of the dye to the protein is approximately in stoichiometry, so the relative amounts of protein can be determined by densitometry. For most SDS and native gels, separated proteins can be simultaneously fixed and stained in the same solution. 

Nonspecific binding of protein to the specimen Use nonimmune serum from same animal species as the secondary antibody to reduce nonspecific binding. A higher concentration of salt in the buffer solutions, such as use of a 1 10 Tris saline solution may also aid in the reduction of nonspecific binding. 

Selectivity is the ability of an assay to measure the analyte of interest in the presence of other constituents in the sample. Because IAs are often performed without sample extraction, they are more prone to matrix interference than are chromatographic methods with extraction. Matrix interference could come from crossreactivity with structurally similar components in the sample, or from nonspecific binding to structurally dissimilar components in the matrix. The results are high background noise, loss of sensitivity, and inaccurate and nonreproducible data. Sometimes, the problem may only occur in a few exceptional patient samples that have structurally similar components such as unknown metabolites, or dissimilar components from samples with hyperlipidemia, hemolysis, complement components, rheumatoid factors, binding proteins, autoantibodies, and heterophilic anti-immunoglobulin Ab. 

Compounds with a molecular weight below 150 or above 500. Very small molecules like benzene are known to bind to proteins in a rather nonspecific manner and at several sites. Very large molecules (like polypeptides) are difficult to optimize subsequently because bioavailability is usually low for compounds with a molecular weight above 500. 

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