Streptavidin, immobilized

Patterning of enzyme monolayers on a solid surface was carried out by photoactivation of immobilized monolayer of caged -biotin derivatives in selected areas. Specific oriented binding of enzyme-avidin conjugates could be readily made to the photoactivated zones [42]. Oriented immobilization of G-protein-coupled receptors on a solid surface was also made possible on a biotinylated surface by first immobilizing streptavidin, followed by the immobilization of biotinylated G-protein-coupled receptor [43]. 

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

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). 

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because the biotin label can be captured or detected using an avidin or streptavidin derivative. For instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled... 

The labeled glycans may be analyzed by cell sorting after staining with fluorescently modified streptavidin. Alternatively, the cells may be lysed and the labeled glycans isolated using immobilized streptavidin. 

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

The 3-D NPH technology was applied to immobilize streptavidin (SA) onto Au-coated surface for surface plasmon resonance imaging (SPRi). By using 3-D NPH method, it was possible to improve the sensitivity of protein-protein interactions drastically comparing to the conventional protein immobilization method. 

Fig. 4 Macromolecular bioconjugates created using bifunctional streptavidins. Here, a biotinylated single-stranded DNA is bound to immobilized streptavidin. A complementary strand covalently attached to streptavidin is hybridized to the immobilized DNA, and a biotinylated protein is bound to the floating streptavidin layer...

Both biotin and strept(avidin) may be covalently attached directly to a variety of surfaces including glass, oxidized silicon, silicon wafers, gold surfaces, filter membranes, and even blood cell membranes using a large variety of methods. Examples of linkers used to immobilize streptavidin or biotin to sulfur-modified silicon surfaces are shown in Fig. 6. Besides direct immobilization, indirect immobilization may occur by binding biotinylated molecules that have been immobilized (Fig. 7). 

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)...

Fig. 3 Scanning electron microscopy (SEM) images of (a) a 150 pm-diameter latex beads spot, (b) a closer view of the latex beads arrangement within a spot (reproduced from [13]) (with permission) (c) immobilized streptavidine-coated beads (2.8 jim) on a biotin modified surface (reproduced from [14]) (with permission), (d) 3D representation of the SEM image of a Sepharose bead trapped at the PDMS/air interface (reproduced from [15]) (with permission)...

Figure 4.3 Standard curves for cytochrome c binding to immobilized aptamer CH6 (squares) and 37-lF (circles). The steady-state signals obtained during injection were corrected for the corresponding signal observed with the control flow cell containing immobilized streptavidin.

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