Nonspecific binding properties

Biotin modification reagents are widely used to attach a biotin group to proteins or other molecules for subsequent use in avidin, streptavidin, or NeutrAvidin separations or assays. Traditional biotin compounds containing aliphatic or other hydrophobic linker arms are discussed in detail in Chapter 11. In this section, the biotin-PEG compounds exclusively are discussed due to their unique hydrophilic properties, which include low nonspecific binding character and low immunogenicity. 

The appropriate definition of nonspecific binding is essential prior to characterization of the kinetic and equilibrium properties of the binding interaction. As a rule, nonspecific binding can be defined using a concentration of the unlabelled ligand that is 100 times its Ka value for the sites of interest. Failure to appropriately define nonspecific binding will invalidate the determination of the binding parameters. 

The final performance of the immobilized NA probes in the hybrid capture of target molecules (sensitivity and discrimination) is strongly dependent upon a number of parameters related to the immobilization process. These include (i) the chemical and physical properties of the surface, since they can influence nonspecific binding of target and nontarget (in mixtures) molecules (ii) the distance between the immobihzed NA probes and the solid surface and the orientation of the immobilized probe (iii) the density of the... 

The isolation of both specific and nonspecific binding proteins on affinity matrices bearing bioactive compounds hinders the identification of drug cellular targets. While solid-phase elution or the competition methods are conventionally used to distinguish between specific and nonspecific receptor-ligand interactions, these approaches are often severely restricted by low ligand solubility and/or slow kinetic dissociation (8). This low solubility of these compounds are not uncommon, since the hydrophobic properties of these compounds are often vital for their bioactivity and/or membrane permeability. 

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