Streptavidin activation with

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.

To make a reference for the 3-D NPH-streptavidin (3-D NPH-SA), the solution including no poly (NAM-co-NAS) was dispensed on the Au-coated slide activated with succinimide esters. The product was referred to as 2-D reference (2-D SA). 

The spotting experiment must be finished within 1 h after preparing streptavidin/poly (NAM-co-NAS) mixed solutions. Because the reaction between the succinimide esters of poly(NAM-co-NAS) and the amino groups of SA molecules and hydrolysis of the succinimide esters of poly (NAM-co-NAS) proceed before printing on substrates in 384-well, 3-D NPH-SA is difficult to be made on Au-coated slide activated with succinimide esters. 

Assessment of protein-associated activity with trichloroacetic acid precipitation a. Dilute a small volume of the pooled radiolabeled streptavidin with saline solution such that 50 pL of the diluted solution has 104—106 cpm. b Add 50 pL of the diluted streptavidin solution to a 12 x 75-mm glass tube, followed by 500 pL of a 0.1% BSA solution in saline, c. For precipitating the proteins, add 500 pL of 10% (w/v) TCA solution in saline. 

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. 

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

Coatings of allyldextran monolayers were carried out on PS chips activated with y -irradiation. In a second step, sodium periodate chemistry was applied to functionalize the dextran layer, followed by the coupling with Streptavidin and/or Neutravidin. The modified PS surfaces are highly hydrophilic with low... 

The well-studied biotin-streptavidin system with its extremely high binding affinity (K 10 M" ) is chosen to illustrate the attributes of these LSPR-based nanoscale affinity biosensors. The biotin-streptavidin system has been studied in great detail by SPR spectroscopy and serves as an excellent model system for the LSPR nanosensor. Streptavidin, a tetrameric protein, can bind up to four biotinylated molecules (i.e. antibodies, inhibitors, nucleic acids, etc.) with minimal impact on its biological activity and, therefore, will provide a ready pathway for extending the analyte accessibility of the LSPR nanobiosensor. 

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). 

These techniques have been used to target, detect, or assay glycoproteins in solution or on cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section 5) (Bayer and Wilchek, 1990 Bayer et al., 1987a, b, 1990) and to form conjugates with glycoproteins. 

Sodium periodate also may affect tryptophan residues in some proteins. The oxidation of tryptophan can result in activity losses if the amino acid is an essential component of the active site. For instance, avidin and streptavidin may be severely inactivated by treatment with periodate, since tryptophan is important in forming the biotin-binding pocket. In addition, many other amino acid residues are susceptible to oxidation by periodate (Chapter 1, Section 1.1). Limiting the time of oxidation is important to restricting oxidation to diol groups while not affecting other protein structures. 

FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

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