Nonspecific binding examples

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

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). 

Fig. 7.16 Saturation isotherm of NO 711 binding to mGATl-membrane fraction as measured in MS binding experiments. One representative example from a series of identical experiments is shown. Total binding of NO 711 ( lOpg protein according to Bradford). Nonspecific binding (o) measured as binding of NO 711 in the presence of 10 mM GABA. Each data point depicts the mean + SEM from triplicate values.

Fig. 10.7 ESI-FTICR mass spectra of three RNA targets at 2.5 pM each screened against 11 compounds at 25 pM each. The percent complexes and one-point values are shown for each ligand complex, (a) An example of a ligand that specifically binds target 2. (b) An example of a ligand that nonspecifically binds to all targets.

The proper choice of an application buffer can help to minimize any nonspecific binding due to undesired sample components. For example, coulombic interactions between solutes and the support can often be decreased by altering the ionic strength and pH of the application buffer. In addition, surfactants and blocking agents (e.g., Triton X-100, Tween-20, bovine serum albumin, and gelatin) may be added to the buffer to prevent nonspecific retention of solutes on the support or affinity ligand. 

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