Avidin amination

Figure 6.1 The Wedekind trifunctional crosslinker can react with amine groups via its p-nitrophenyl ester to form amide bond linkages. The phenyl azide group then can be photoactivated with UV light to generate covalent bond formation with a second molecule. The biotin side chain provides binding capability with avidin or streptavidin probes.

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

NHS-iminobiotin can be used to label amine-containing molecules with an iminobiotin tag, providing reversible-binding potential with avidin or streptavidin. The NHS ester reacts with proteins and other amine-containing molecules to create stable amide bond derivatives (Figure 11.6). An iminobiotinylated molecule then can be used to target and purify other... 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

Protocol for the Conjugation of (Strept)avidin with Ferritin Using Reductive Amination... 

Conjugation of HRP by reductive amination can be done by oxidizing the carbohydrate on the enzyme and subsequently coupling to the amines on (strept)avidin (Figure 23.5). 

Figure 23.5 Oxidation of the polysaccharide components of HRP produces reactive aldehyde groups. Conjugation to avidin then may be done by reductive amination.

Figure 23.8 AMCA-NHS reacts with the amine groups of avidin (or (strept)avidin) to produce amide bonds.

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

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