Avidin reaction with biotin

Reaction with avidin-labeled alkaline phosphatase (AP)-biotin conjugate... 

The increased length of this spacer (24.7 A) provides more efficient interaction potential with avidin or streptavidin probes, possibly increasing the sensitivity of assay systems. The reactions of biotin-LC-hydrazide are identical to those of biotin-hydrazide. 

Block nonspecific staining due to cross reaction with endogenous avidin or biotin by incubation with avidin solution followed by biotin solution, both for 20 min (use Vectorstain kit, as directed, Vector Labs, Burlingame, CA). 

The avidin-biotin technique is especially useful in double-labeling experiments. This can be done using two different enzymes in the final step, combined with two different andbody species (monoclonal and/ or polyclonal). One of the andbodies can be biodnylated and the other immunochemical reaction can be based on a different procedure (e.g., employing an antibody-enzyme conjugate). Alternatively, both antibodies can be biotinylated and the secondary reaction with the respective avidin-enzyme conjugate can be performed on different sides of an impermeant (e.g., plastic-embedded) tissue section. 

By itself, biotin is not a detectable label. It is used as a label because a subsequent irreversible reaction with the proteins avidin or streptavidin (K > 1012 M 1) can be used for ultrasensitive detection, if these proteins have been labeled with an enzyme and an activity stain is used. Conjugates of these proteins with alkaline... 

Fig. 2.4. Competitive, homogeneous EIA. The symbol E represents enzyme, A antigen or hapten, S and CO substrate and enzyme cofactor, and Av and B avidin and biotin. Enzymes indicated with have modulated (increased or decreased) activities. In (a) hapten is conjugated to the enzyme and reaction with the antibody (shaded structure) modulates the enzyme activity. This interaction is, however, prevented if antigen is present in the sample. Similar principles apply if substrate (b) or cofactor (c) are conjugated with the antigen. In (d) antibody is directly linked to the enzyme, and the reaction of this antibody, particularly with high-molecular weight antigens, will block the active site of the enzyme. In (e) antibody, directed to haptens labeled to avidin, prevents avidin from inhibiting the biotin-containing enzyme, unless antibody is neutralized by the same hapten present in the sample.

A simple and economical way to obtain complexes of optimum activity consists of the use of small column of iminobiotin saturated with avidin according to Section 3.2.1.3 (crude extract from egg whites can be used directly). One of the binding sites of avidin is occupied by the iminobiotin while the other remains free for reaction with the subsequently added biotinylated POase. Avidin is then added to saturate additional free biotin groups on POase. Complexes are then eluted from the column with 50 mM ammonium acetate (pH 4.0). 

There have been many reports that described protein composites containing metal complexes [1-3, 5, 6], Three different approaches for the incorporation of synthetic metal complexes into protein cavities have been reported (i) modification of natural snbstrates, (ii) covalent anchoring, and (iii) non-covalent insertion. For example, Whiteside et al. constructed artificial metaUoenzymes by the conjugation of a Rh diphosphine complex with biotin, which strongly binds to avidin [8], Ward et al. have improved this method to increase the reaction activities [25]. They optimized the reaction conditions by screening the structures of metal complexes and protein environments. Finally, optimized composites catalyzed asymmetric hydrogenation with np to 97% ee (Fig. 2a) [2, 3, 26],... 

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