Solid surface methods proteins

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

An ordered antibody array has also been assembled on the solid surface by a combination of Langmuir Blodgett (LB) film method and self-assembling method. An ordered monolayer of protein A is deposited on the solid surface by LB method, which is followed by self-assembling of antibody. Individual antigen molecules which are complexed with the antibody array have been quantitated selectively by atomic force microscopy (AFM). 

Protein A is a cell-wall protein of Staphylococcus aureus with a molecular weight of 42,000. Since protein A binds specifically to the Fc part of IgG from various animals, it has been widely used in immunoassay and affinity chromatography. We found that protein A could be spread over the water surface to form a monolayer membrane by the LB method [21]. On the basis of this finding, an antibody array on the solid surface can be obtained by the following two steps. The first step is fabrication of an ordered protein A array on the solid surface by the LB method. The second step is self assembly of antibody molecules on the protein A array by biospecific affinity between protein A and the Fc of IgG as shown in Fig.34. 

Fluorescence is also a powerful tool for investigating the structure and dynamics of matter or living systems at a molecular or supramolecular level. Polymers, solutions of surfactants, solid surfaces, biological membranes, proteins, nucleic acids and living cells are well-known examples of systems in which estimates of local parameters such as polarity, fluidity, order, molecular mobility and electrical potential is possible by means of fluorescent molecules playing the role of probes. The latter can be intrinsic or introduced on purpose. The high sensitivity of fluo-rimetric methods in conjunction with the specificity of the response of probes to their microenvironment contribute towards the success of this approach. Another factor is the ability of probes to provide information on dynamics of fast phenomena and/or the structural parameters of the system under study. 

Vroman has shown by antibody methods that plasma interactions with solid surfaces result in a hierarchial adsorption process 98). The high concentration proteins dominate the surface at short times due to the higher collision rates. As time passes... 

Rideal and his collaborators found that solid particles of protein spread very rapidly on a clean surface their method of spreading is to coat a quartz fibre, first covered with paraffin wax, with a little protein this is dipped into the water surface, and the amount of protein passing into the surface ascertained by weighing the fibre on a simple micro-balance.1 By this method somewhat larger areas can sometimes be obtained, and films formed from proteins which cannot be made to form homogeneous films when spread by Gorter s technique and in certain cases the average thickness of the fully spread film is only about 3 A. 

Fig. 7 Approaches for linking macromolecules to solid surfaces using biotin-strept(avidin) systems. Either biotin or streptavidin may be directly linked to a surface (e.g. Fig. 6). There are a large number of biotin derivatives with different spacer arms (e.g. Fig. 3) that may be used for immobilization. The choice of a specific biotin depends on which active group occurs on the native or derivatized surface. Attachment of strept(avidin) may be done with methods used with other proteins. Note that the deposition of a biotinylated molecules to a surface may also be used (e.g. biotinylated small molecules or large molecules like BSA) to link strept(avidin) to a surface. A DNA covalently attached at one end (3 end) can have a functional biotin at the other end that could bind to strept(avidin) or be covalently linked to streptavidin (see Fig. 4). Single-stranded DNA bound to immobilized streptavidin is available for hybridization to its complementary sequence (See Fig. 4)...

The strong interaction with the solid surface sometimes compensates for a lack of self-assembling ability for some components (Fig. 4.36). Molecules whose structures are much different to those of typical amphiphiles can be used in this method. In addition, immobilizations of dyes, proteins and nucleic acid are possible using this self-assembled monolayer technique. These self-assembled monolayers have great potential for a wide range of applications. 

Figure 1 shows the physicochemical surface methods used extensively in our laboratory to assess the interfacial structure and properties of predominantly protein substrates like skin, collagen, and living cell surfaces and also to assess the initial sequence of events at clean solid substrates upon their exposure to blood, saliva, and sea water. 

In general, the site-specific immobilization methods described above are especially effective when the protein recognition site on ligand molecules is known or can be predicted. Small molecules immobilized on solid surfaces through a specific functional group uniformly present a certain area of their molecular surfaces, and thus the interaction with proteins that recognize this surface can be analyzed in a highly sensitive and quantitative manner. However, except in the case of certain protein-reactive small molecules [53], it is difficult to predict the... 

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