Avidin, detection

Based apparently on the same principle as described above, suitable but rather expensive kits exploiting monovalent Fab fragments to block endogenous tissue immunoglobulins have also been developed commercially, such as Mouse-on-Mouse (M.O.M. ) Kits by Vector Laboratories (http //www.vectorlabs.com/). Three Vector M.O.M. kits are available. These kits use the same blocking technology and biotin-avidin detection format, but offer a choice of using either an enzyme-based or fluorescent-based visualization method. 

Wood G, et al. Suppression of Endogenous Avidin-Binding Activity in Tissues and Its Relevance to Biotin-Avidin Detection Systems. Journal of Histochemistry and Cytochemistry 2981 29 1196-204. 

Probe Detection. There are a number of different protocols for the detection of hybridized probe and these are summarized in Table 2. The choice of detection system will be dictated by your choice of probe label, (biotin or dig), and whether you are carrying out single or double hybridizations. For single hybridizations with biotin-labeled probes, we favor the avidin-detection protocol (see Subheading 3.8.1.), and for double hybridizations with biotin- and dig-labeled probes simultaneously, the avidin/anti-dig protocol (see Subheading 3.8.3.). 

Metagenomics has been applied to the search for novel genes encoding the synthesis of vitamins such as biotin and vitamin C [47,76]. Seven cosmids were detected in metagenomic libraries obtained after avidin enrichment of environmental samples. The highest levels of biotin production in this study were detected in a cosmid... 

Although biotin-fluorophores have been described in the detection of biomolecules, namely proteins, peptides, or DNA, most of these loose part of their fluorescence when binding to the tetrameric proteins avidin and streptavidin. 

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. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

To reduce the hydrazone bonds to more stable linkages, cool the solution to 4°C and add an equal volume of 30 mM sodium cyanoborohydride in PBS. Incubate for 40 min. Note If the presence of a reducing agent is detrimental to protein activity, eliminate this step. In most cases, the hydrazone linkage is stable enough for avidin-biotin detection experiments. 

Psoralen-PEOj-Biotin has been used to label double-stranded DNA for detection using (strept)avidin reagents (Henriksen et al., 1991 Wygrecka et al., 2007). The psoralen photoreactive group provides better insertion yields than typical phenyl azide-based systems, such as the standard photobiotin probe discussed previously in this section. 

Block nonspecific sites on the membrane with an appropriate blocking component (such as BSA) and detect the biotinylated proteins using an avidin or streptavidin conjugate. 

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because the biotin label can be captured or detected using an avidin or streptavidin derivative. For instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled... 

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.

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