Nonspecific binding sources

Behnke, O., Ammitzboll, T., lessen, H, Klokker, M., Nilhausen, K., Tranum-Jensen, H., and Olsen, L (1986) Nonspecific binding of protein-stabilized gold sols as a source of error in immunocytochemistry. Eur J Cell. Biol 41,326-338... 

In some cases, the positively charged properties of egg-white avidin provide an advantage for its use in lieu of streptavidin. This is particularly the case when high levels of derivatization (for example in the synthesis of a fluorescent derivative of avidin) is required. Thus, the high number of free lysines in avidin furnishes an excellent medium for extensive derivatization with simultaneous reduction of the pi to an acceptable level. The use of nonglycosylated avidin for such purposes would counteract the other major source of nonspecific binding, i.e., saccharide-specific interactions. 

The CoA-fortlfled microsomal enzyme system was inhibited by SKF 525-A (B-diethylaminoethyl-diphenylpropyl acetate) and by 2,4-dlchloro-6-phenylphenoxyethylamine HBr (DPEA) in a dose-dependent fashion (28). Both are known inhibitors of the classical hepatic microsomal mixed-function oxidase system. This inhibition has been suggested to Involve a nonspecific binding to liver microsomal proteins and/or phospholipids (18.19). Binding to phospholipids would support the hypothesis proposed by Swell and co-workers (10) that fatty acyl CoA derivatives were not necessarily the only immediate source for the fatty acid moieties of the conjugates that phospholipids may act as the fatty acids reservoir in the CoA-fortified enzyme system. However, it is also known that cholesterol esterification is very sensitive to the fluidity of the microsomal membrane (12). The reduced conjugation produced when... 

Fig. 5.1 Sources of nonspecific binding. Incubation with antibodies should have only antibody binding to the correct antigen. Nonspecific binding can result from charged groups that bind proteins including antibodies. The tissue can have Fc receptors that bind to the Fc region of any antibody. Some tissues can have exposed antibodies...

Oxygen-derived free radicals are also a major source of DNA damage. Approximately 20 types of oxidatively altered DNA molecules have been identified. The nonspecific binding of Fe to DNA facilitates localized production of the hydroxyl radical, which can cause base alterations in the DNA (Fig. 24.9). It also can attack the deoxyribose backbone and cause strand breaks. This DNA damage can be repaired to some extent by the cell (see Chapter 12), or minimized by apoptosis of the cell. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

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