Streptavidin 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... 

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.

Surface immobilization of the capture molecules follows standard procedures that are commonly practiced in many biosensor applications and some are discussed in the previous section. Layers of carboxymethyl dextran. Protein A or Protein G, streptavidin-coated surface, or EDC [N-ethyl-N-(diethylaminopropyl) carbidimide]/NHS (N-hydroxysuccmimide)-based amine coupling through amide bond are used for protein (antibody, receptor, etc.) cross-linking. 

Conjugation of HRP by reductive amination can be done by oxidizing the carbohydrate on the enzyme and subsequently coupling to the amines on avidin or streptavidin (Fig. 364). 

Protocol for the Preparation of Avidin—HRP or Streptavidin—HRP by Reductive Amination... 

Fluorescein isothiocyanate (FITC) is the most common fluorescent label used to modify proteins and other biomolecules (Chapter 8, section 1.1). The isothiocyanate group reacts with amines in protein molecules to form a stable thiourea linkage (Fig. 365). Avidin or streptavidin may be tagged with this reagent to yield highly fluorescent... 

Figure 367 AMCA—NHS reacts with the amine groups of avidin (or streptavidin) to produce amide bonds.

A wide range of immobilization chemistries are commercially available in conjunction with Sepharose beads. We have investigated a limited subset of these possibilities which include direct, nonoriented immobilization via Schiff s base chemistry, oriented nonco-valent immobilization via immobilized metal affinity chromatography resins and oriented noncovalent immobilization via biotin-streptavidin binding. At present we favor direct, covalent attachment of proteins via primary amines since it is highly efficient (typically better than 85% yield), minimizes leaching and provides the best NMR results (Figure 6.2). 

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