Affinity-interaction methods

Zhou, R. Friesner, R.A. Ghosh, A. Rizzo, R.C. Jorgensen, W.L. Levy, R.M., New linear interaction method for binding affinity calculations using a continuum solvent model, J. Phys. Chem. B 2001,105,10388-10397... 

Recently, Vasilescu et al. demonstrated the use of formaldehyde to preserve protein interactions in vivo followed by immunoaffinity purification of a targeted complex, cross-link reversal via heating at 95°C, separation by SDS-PAGE, and identification of bands by LC-MS/MS.7 Tagwerker et al. utilized formaldehyde cross-linking in conjunction with a novel tag-based affinity purification method.36... 

For immunoprecipitations from native tissues, one requires antibodies directed against both the fish and the bait proteins. Further, these antibodies should not bind to epitopes within the putative protein-protein BDs. It is technically difficult to determine low affinity or transient association among proteins by immunoprecipitation because low-affinity interactions may be lost by washing immune pellets to remove nonspecifically bound proteins. Also, one cannot manipulate protein concentrations to favor protein association as one can in a pull-down assay. Under these circumstances, probably the best method to use would be FRFT. 

Many papers have described methodologies useful for characterizing ligand-protein interactions (63-67). However, among these methods, ACE has been the subject of much attention recently for the evaluation of affinity interactions because of both its high resolution and the extremely small amounts of sample required. 

Proteomics can make a key contribution in the identification of interacting protein partners and create a protein-protein interaction map of the cell (Lamond and Mann, 1997 Neubaer et ah, 1997 Blackstock and Weir, 1999 Link et ah, 1999). This would be of immense value to understanding the biology of the cell and can be further exploited for drug development. The simplest way to study such interaction is to purify the entire multiprotein complex by affinity-based methods. The members of the multiprotein complex can then be identified by mass spectrometry. 

Au NPs functionalized with biomolecules can be synthesized using different methods, depending on factors inducing the interactions promoted between the nanoparticle and the biomolecule. These interactions can be classified as electrostatic adsorption, chemisorption and covalent binding and, finally, specific affinity interactions. Some examples are given in the following paragraphs (Scheme 3.23). 

Electrochemical methods of detection affinity interactions at the surfaces are rather effective due to their relative simplicity and low cost. Amperometric aptasensor based on sandwich assay was proposed by Ikebukuro et al. [42]. They used two aptamers selective to thrombin. 

SPR method is currently well established and used in analysis of affinity interactions. The commercial instruments produced by Biacore AB or Texas Instruments are available in the market. Substantial progress in SPR instrumentation is connected also with the possibility to detect simultaneously the affinity interaction from several surfaces [72], This approach has been proved equally effective for detection of thrombin-DNA aptamer interactions [36]. 

Attachment via affinity interactions. These methods are based on the high affinity of Fc receptors, such as protein A (PrA), protein G (PrG) or recombinant protein A/G, for the amino groups in the Fc region of antibodies. In these procedures, the Fc receptor is immobilized on a solid support and is the key element for the oriented immobilization of the antibody (Fig. 5b). However, not all Fc receptors have the same affinity to all IgG isotypes. PrA has been successfully used to bind the Fc portion of antibodies from many mammalian species, except for goat, sheep, cow and horse. PrG has some advantages over protein A, as it reacts with more IgG isotypes and to a lower extent with other immunoglobulins, such as human IgM and IgA. How-... 

Probably the best means of protein attachment is through highly specific affinity interactions (e.g., streptavidin-biotin or His-tag-nickel-chelates) [111]. Proteins fused with a high-affinity tag, at their amino or carboxyl terminus, are finked to the surface of the chip via this tag, and hence all the attached proteins should orient uniformly away from the surface. Using this method, immobilized proteins are more likely to remain in their native conformation, while the analytes have easier access to the active sites of the proteins. 

Affinity Chromatography was initially defined as a method based on specific and reversible molecular interactions between biologically active substances. However, the method has extended to non-biological stationary phases, such as metal-chelate complexes. This technique is used for separation and purification of proteins and other biologic materials, such as viruses and cells.47,48 A survey of various stationary phases and affinity interactions is given in figure 8.2. 

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