Linkage through immobilized protein

For immobilization studies to date, two distinct modes of immobilization have been used. The first utilizes nonspecific covalent bonding to CNBr-activated Sepharose via primary amino groups on the antibody molecule. Since there are many of these available on the antibody, this is expected to result in random orientation of antibody molecules on the support. The other method involves linkage through immobilized protein A, a protein which binds immunoglobulins in the structural F portion of the molecule. 

Chen et al. (2007) have developed a nanoinjector that injects compounds immobilized on MWNT-atomic force microscopy (AFM) tips into the cells. First, a MWNT-AFM tip was fabricated from a normal AFM tip with an MWNT on one end. Next, a compound of interest was immobilized on the MWNT-AFM tip through a disulfide bond linkage. After MWNT-AFM tip was tapped on the cell, the cantilever was further lowered and the MWNT nanoneedle then penetrated the membrane. Once inside the cell, the disulfide linkage was broken under the cells reducing environment and the compound of interest was released inside the cell. The MWNT-AFM tip was then removed from the cell. In this study, protein was... 

As schematically depicted in Figure 5, two different routes are available for immobilizing biotin-labeled enzymes on the support through avidin-biotin complexation. The first procedure employs the biotin-modified surface on which biotin-labeled enzymes are immobilized through avidin as binder protein. For this procedure, the covalent linkage of biotin onto the surface of a carbon electrode and the preparation of biotin-labeled lipid bilayer on electrode have been studied. An alternative way involves the direct modification of an electrode surface with avidin. If avidin could be immobilized directly without loss of the binding activity to biotin, biotin-labeled enzymes could be loaded more easily on the electrode surface. 

Nucleic acids have been linked to a variety of surfaces including polystyrene beads, glass, silicon, gold, and even cells. Immobilization of nucleic acids may occur through a number of covalent linkages that are the subject of other chapters. Strept(avidin) may then be bound to a surface through a biotinylated nucleic acid linker. Alternatively, strept(avidin) may be linked to a surface directly with methods used for other proteins. This chapter will describe the biotin-strept(avidin) system and focus on the use of the biotin-strept(avidin) to link nucleic acids to surfaces. 

Wu P, Brand L. N-terminal modification of proteins for fluorescence measurements. Methods Enzymol. 1997 278 321-330. Gilmore JM, Scheck RA, Esser-Kahn AP, Joshi NS, Erancis MB. N-terminal protein modification through a biomimetic transamination reaction. Angew. Chem. Int. Ed. 2006 45 5307-5311. Christman KL, Broyer RM, Tolstyka ZP, Maynard HD. Site-specific protein immobilization through N-terminal oxime linkages. J. Mater. Chem. 2007 17 2021-2027. 

One advantage of SPRi is the fact that no label is necessary, although the targets are immobilized on a support, often through covalent linkage. The interaction of proteins with any other classes of compounds, such as lipids, small molecules. 

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