Streptavidin fluorescent labeling

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). 

Figure 10.19 ATP-fueled rotation of fluorescently labeled actin that is attached to the y subunit of the F,-ATPase by a streptavidin-biotin linker. The /3 subunits are immobilized on a microscope slide. [Modified from H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Nature 386,299 (1997).]...

N-terminals of the /3 subunits. At the other end, a cysteine residue was introduced into the exposed tip of the y subunit, which was coupled to biotin, and then attached to a fluorescently labeled actin filament via a streptavidin linker. The ATP-dependent anticlockwise rotation of the 1- to 3-p.m-long actin filaments was seen in a fluorescence microscope.94 Smaller probes show that the rotation is consistent with the turnover of ATP by the FrATPase, which is consistent with a three-step motor.95,96 This implies that ATP synthesis requires that the y subunit be cranked in a clockwise direction by a rotary motor in F,. 

If the ligand protein is fluorescently labeled, resuspend the cells in 10 pL PBS and subject directly to FACS. If the ligand protein is biotinylated (protocol 2), resuspend the cells in 10 pL PBS containing streptavidin, R-phycoerythrin conjugate (1 10 dilution in PBS). Incubate on ice again for 10 min. 

Fluorescein isothiocyanate (FITC) is the most common fluorescent label used to modify proteins and other biomolecules (Chapter 8, section 1.1). The isothiocyanate group reacts with amines in protein molecules to form a stable thiourea linkage (Fig. 365). Avidin or streptavidin may be tagged with this reagent to yield highly fluorescent... 

Fig. 6. Measurement of the relative amount of ligand bound to each protein in the array. (A) Schematic of on-chip binding assay in which a fluorescently labeled interaction partner binds to the functional, arrayed protein immobilized to the streptavidin-coated surface via the biotinylated BCCP tag. (B) p53 protein function microarray probed with Cy3-labeled GADD45 duplex oligo. Quantification of the signal intensity from each spot allows the effect of polymorphic and functional variation on the DNA binding function of p53 to be determined.

FIGURE 6.35 Schematic of the experimental protocol for streptavidin affinity chromatography. (1) The channel is initially filled at room temperature with a suspension of biotinylated, PNIPAAm-coated beads (100 nm). (2) The temperature in the channel is then raised to 37°C, and the beads aggregate and adhere to the channel walls. (3) Buffer is then pumped through the channel (the presence of flow is indicated in this schematic by an arrow), washing out any unbound beads. (4) A fluorescently labeled streptavidin sample (2.5 pM) is then introduced into the flow stream. (5) Streptavidin binds to the beads, and any unbound streptavidin is washed out of the channel. (6) Finally, the temperature is reduced to room temperature, leading to the breakup of the bead aggregates. Beads, bound to labeled streptavidin, elute from the channel [203], Reprinted with permission from the American Chemical Society. 

In 2001, Van Orden et al. reported the first biologically relevant FRET investigation using QDs as energy donors.83 They conjugated biotinylated bovine serum albumin (bBSA) to water-soluble CdSe-ZnS-TA QDs via a thiol linkage with 2-iminothiolane. The acceptor was streptavidin covalently labeled with tetra-methylrhodamine (TMR). Once the avidin-biotin interaction took place, a decrease in the QD luminescence and an increase in the TMR luminescence were observed, which was attributed to FRET between QD and TMR. Although no time-resolved fluorescence experiments were carried out and no estimates for the separation distance... 

Fig. 9 c Surface was flooded with fluorescently labeled streptavidin... 

To demonstrate that STORM can indeed resolve nearby fluorescent molecules with sub-diffraction-limit resolution, we first engineered samples with known relative positions of the fluorescent labels - double-stranded DNA labeled with two Cy3-Cy5 pairs separated by a well-defined number (135) of base pairs, corresponding to an inter-CyS distance of 46 nm along the contour of DNA [4]. The DNA strands were immobilized in a flat configuration to a quartz slide through multiple biotin-streptavidin linkages. The two Cy5 dyes were turned on and off, repetitively, and the image sequence was analyzed to determine the positions of individual activated Cy5 dye. We then constructed... 

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