Avidin activation with

A variation of the above method can be used, wherein the enzyme is first activated with SMCC and conjugated to a thiolated (strept)avidin molecule. This approach probably is the most common way of preparing (strept)avidin-enzyme conjugates, and since the preactivated enzymes are readily available (Thermo Fisher), it also may be the easiest. 

Protocol for the Conjugation of SMCC-Activated Enzymes with Thiolated (Strept)avidin Activation of Enzyme with Sulfo-SMCC... 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

The following protocol describes the activation of avidin or streptavidin with sulfo-SMCC and its subsequent conjugation with an enzyme modified to contain sulfhydryls using SATA (Chapter 1, Section 4.1). A method for the opposite approach, wherein the enzyme is activated with SMCC and the avidin component is thiolated, is presented immediately after this protocol. This strategy may be the most common approach to forming these conjugates (Fig. 363). In addition, since there are enzymes commercially available that are preactivated with SMCC (Pierce), their use may be the easiest solution. 

The presence of this artifact poses a distinct risk of its being interpreted as positive staining, as the artifact can be intense and may be precisely located in the cells of interest with a clean background. It is known that liver and kidney can retain high amounts of retrievable biotin-avidin activity in neoplasms. The need for adequate controls or biotinblocking procedures is obvious when histochemical or immunohistochemical procedures are used. Negative controls facilitate identification of such nonspecific staining. 

Avidin is a tetrameric protein and binds 4 mol of biotin per tetramer it also binds iV-carboxybiotin with a somewhat lower affinity. The unit of avidin activity is that amount which will bind 1 /rg (4.09 nmol) of biotin commercially available avidin has an activity of 10 to 15 units per mg of protein. 

This technique has been developed by Aizawa and coworkers. The goal is to build a convenient and specific detector using an enzymatic activity as signal. In the case of biotin determination, avidin coupled with catalase is bound to a membrane bearing covalently linked HABA residues. Addition of biotin destroys quantitatively the HABA-avidin-catalase complex. Washing the membrane and measurement of the remaining catalase activity afforded a sensitive (0.5 ng) and convenient titration of biotin (95). 

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of HCV in serum by Skladal et al. [66]. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). 

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

To clarify the characteristics of AMDase, the effects of some additives were examined using phenylmalonic acid as the representative substrate. The addihon of ATP and coenzyme A did not enhance the rate of the reaction, different from the case of malonyl-CoA decarboxylase and others in those, ATP and substrate acid form a mixed anhydride, which in turn reacts with coenzyme A to form a thiol ester of the substrate. In the present case, as both ATP and CoA-SH had no effect, the mechanism of the reaction will be totally different from the ordinary one described above. It is well estabhshed that avidin is a potent inhibitor of the formation of the biotin-enzyme complex. In the case of AMDase, addition of avidin has no influence on the enzyme activity, indicating that AMDase is not a biotin enzyme. 

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