Biotinylated fluorescein

Confocal laser scanning fluorescence microscopy was used to study the exposure of the avidin-specific binding sites in the Av-GEB platform by the immobilization of a small and flexible biotinylated fluorescein molecule as a fluorescence marker. Fluorescence microscopy thus confirms that Av-GEB platform exposes active binding sites for biotin, acting as affinity matrix (Fig. 21.2B). After use, the electrode surface can be renewed by a simple polishing procedure for further uses, highlighting a clear advantage of this new material with respect to surface-modified approaches such as classical biosensors and other common... 

Fig. 21.2. (A) Schematic representation of the electrochemical DNA biosensing procedures based on Av-GEB. (B) Confocal laser scanning fluorescence microphotograph of Av-GEB transducers submitted to (i) non-biotinylated fluorescein (background adsorption) and (ii) 80 pmol of biotinylated fluorescein. Laser excitation was at 568 nm. Voltage 352 V (more details in Zacco et at., [65]).

Scheme 1.21. Protein assay operation in the presence of target Streptavidin, the biotin moiety of Fl-B specifically associates with Streptavidin and the fluorescein moiety is buried in the adjacent vacant-binding sites. This makes fluorescein far away from P4 and inefficient FRET between them occurs (A). In the presence of nonspecific target, the strong electrostatic interactions between cationic P4 and negatively charged biotinylated fluorescein (Fl-B) result in efficient FRET from P4 to fluorescein (B)...

Proteomics In MIcrotIuldIc Devices, Rgure 4 (a) Use of hydrodynamic flow fo create a microfluidic stream and direct it to specific areas of a larger channel, (b) The flow system is used to deposit biotinylated fluorescein in a grid pattern. The direction of flow during deposition is indicated by arrows, (c) Quantitation of C-reactive protein (CRP) in whole blood. Samples of blood were spiked with CRP and the microstream directed across a surface coated with anti-CRP before exposure with a Cy3-labeled secondary antibody. Reprinted from [24] under open access agreement with BioMed Central. 

Finally, the ability of avidin/Ag nanoparticle LbL films to act as smart SERS/SERRS substrates was tested. A film composed of 14 bilayers of avidin and Ag nanoparticles was inunersed into a lO M solution of biotinylated fluorescein (biotin-4-fluorescein) for 30 min, while another was immersed into a solution of untagged fluorescein with equal concentration, for an equal period of time. Upon removal, both were thoroughly rinsed with water, dried, and SERRS spectra were recorded from them using 514 nm excitation, it was found that the spectra recorded from the two samples were essentially the same in terms of band frequencies, widths, and relative intensities. (The B4F spectrum is shown... 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

The biochemical MS assay performance was studied for various biotin derivatives, such as biotin [m/z 245), N-biotinyl-6-aminocaproic acid hydrazide (m/z 372), biotin-hydrazide (m/z 259), N-biotinyl-L-lysine (m/z 373) and biotin-N-succinimi-dylester m/z 342). These five different bioactive compounds were consecutively injected into the biochemical MS assay. Figure 5.12 shows triplicate injections in the biochemical MS-based system of the different active compounds. Each compound binds to streptavidin, hence the MS responses of peaks of the reporter ligand (fluorescein-biotin, m/z 390) are similar. The use of SIM allows specific components to be selected and monitored, e.g. protonated molecule of the biotin derivatives. In this case, no peaks were observed for biotin-N-succinimidylester (m/z 342), because under the applied conditions fragmentation occurred to m/z 245. In combination with full-scan MS measurements, the molecular mass of active compounds can be determined simultaneously to the biochemical measurement. 

Fig. 5.12 On-line continuous-flow monitoring of bioactive compounds using fluorescein-biotin/streptavidin assay. MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Triplicate injections of (a) biotin-N-succinimidyl ester (m/z 342), (b) N-biotinyl-L-lysine (m/z 373),...

While the binding of aminoglycosides to the RRE provides a proof of principle, their affinity and, in particular, selectivity traits need to be improved for true therapeutic utility. To facilitate the discovery of potent and selective RRE binders, we developed a solid-phase assay. The components of this assembly include (a) insoluble agarose beads (or microtiter plates) covalently modified with streptavidin, (b) a biotinylated RRE fragment, and (c) a fluorescein-labeled Rev fragment (RevFl). Assembly of the three components generates an immobilized ternary complex whereby the biotinylated RRE binds to the beaded... 

The effects of CVS on the numbers of T-cell subsets were examined on day 9 at the time of tumor i.v. rechallenging, Fig. (6)-Protocol B. Cells were stained with fluorescein 5-isothiocyanate (FITC)-, phycoerythrin (PE)-conjugated and/or biotinylated mAb and were additionally stained with RED-613-streptavidin for flow-cytometric three-color analysis. 

The cells and nuclei (0.5-1.0 X 106) are aliquoted into polystyrene tubes. Approximately 10 jjlI of normal horse serum (Vector Laboratories, Burlingame, CA) is added to block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1 25 dilution for 1 hr at 37°C. Aliquots are stained with 10 p,l of fluorescein isothiocyanate (FITC)-conjugated strepta-vidin (Dako buffer containing 0.1% Triton X-100). 

Forms of test article 47% Unconjugated 15% Biotinylated 34% Fluoresceinated... 

In addition, two different control experiments were also run to confirm the efficacy of the MAMEF assays. Figure 7.5C shows that no fluorescence emission intensity was detectable from the control assay, where biotinylated-BSA is omitted from the assay, which was run at room temperature for 30 minutes. When the identical control assay was run with microwave heating, once again there was little/no fluorescence emission. These two control experiments nicely demonstrated that the non-specific binding of the fluorescein-labeled streptavidin to the surface was minimal. 

The gold nanoparticle/polyelectrolyte coated latex particles are then conjugated with biotin molecules through a layer of biotinylated poly(allylamine hydrochloride) that is deposited on the particle surface before biotin binding. Fluorescein isothiocyanate labled anti-biotin immunoglobulin (FITC-anti-biotin IgG)... 

Some biodnylated andbodies will not react with some secondary reagents. If this is the case (which can only be established by experiment), it is possible to use one biotinylated primary antibody together with a second, unconjugated, primary antibody and a fluorescein-labeled and-Ig. The cells are incubated with the biotinylated antibody followed by streptavidin-PE together with the second primary monoclonal antibody, followed by an andmouse Ig conjugated to fluorescein. 

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