Proteins avidin-biotin complex

The avidin-biotin complex, known for its extremely high affinity (Green, 1975), has been studied experimentally more extensively than most other protein-ligand systems. The adhesion forces between avidin and biotin have been measured directly by AFM experiments (Florin et al., 1994 Moy et al., 1994b Moy et al., 1994a). SMD simulations were performed on the entire tetramer of avidin with four biotins bound to investigate the microscopic detail of nnbinding of biotin from avidin (Izrailev et al., 1997). 

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. 

Figure 10. Three dimensional protein multilayer based on avidin-biotin complexation.

The following procedure is based on the Vectastain ABC kit from Vector (suppliers appendix). It uses an avidin-biotin complex to attach horseradish peroxidase (HRP) or alkaline phosphatase (AP) to the biotinylated secondary antibody. Avidin-biotin systems are capable of extremely high sensitivity because multiple reporter enzymes are bound to each secondary antibody. In addition, the detergent Tween 20 is a popular alternative to protein blocking agents when using nitrocellulose or PVDF membranes. 

A primary example of biological binding immobilization is the avidin-biotin complex. Biotin (vitamin H) is a low-molecular-weight cofactor distributed ubiquitously in cells. Biotin specifically binds to avidin, an egg white protein, or to strepavidin, a similar protein which occurs in Streptomyces sp. 

The procedure begins with the standard ABC protocol, where biotin-labeled 2° antibody is bound with the avidin-biotin complex (ABC). This adds a significant number of HRP molecules for each L antibody. To start the TSA method, incubate tissue with biotin-labeled tyramide with H2O2 the HRP enzymes generate activated tyramide (Fig. 7.10). The activated tyramide then binds to the available tyrosine amino acids on cellular proteins near the antigen, on the antibodies, and even on... 

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