Fluorescent Probes (Biomolecule)

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Fluorescent Probes for Labeling Biomolecules and Their Applications

Fluorescent probes that can selectively label specific biomolecules can be used to visualize the biomolecules, as well as to study their dynamics and activity. Fluorescent probes specific for biomolecules and their applications are described below.

Visualizing Specific Biomolecules

Well-known examples of fluorescent probes for biomolecules are those that specifically bind to nucleic acids (DNA/RNA). Typical staining reagents include DAPI, Hoechst 33258, and Hoechst 33342. The fluorescence intensity of these fluorescent dyes increases when they bind to the minor groove of double-stranded DNA, enabling specific staining of double-stranded DNA. Acridine orange (AO) is incorporated into double-stranded nucleic acids at a ratio of one per three base pairs, and into single-stranded nucleic acids at a maximum of one per base . Because of this difference, complexes with double-stranded nucleic acids emit fluorescence at 520 nm, while complexes with single-stranded nucleic acids emit fluorescence at 620-650 nm, making it possible to distinguish between double- and single-stranded nucleic acids.

Labeled phalloidin is often used to visualize actin filaments. Phalloidin is a peptide found in the death cap mushroom and other fungi. It binds specifically and strongly to filamentous actin. In contrast, anti-actin antibodies also recognize actin monomers. For staining actin filaments, use of phalloidin can suppress the signal derived from monomers.

Visualizing the Dynamics and Activity of Biomolecules

For example, 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) and dihydroethidium (DHE) are commonly used for the detection of reactive oxygen species (ROS). These fluorescent probes react with ROS and change from reduced form to oxidized form, resulting in emission of fluorescence or alteration of the fluorescence wavelength. DCFH-DA and DHE, however, have low specificity for the type of ROS. Fujifilm Wako’s BES probe is a fluorescent probe with a high selectivity for hydrogen peroxide and superoxide.

Enzyme activity can also be measured using fluorescent probes. For the typical reporter β-galactosidase (β-gal), a colorimetric reagent such as X-Gal is usually used, but the use of a fluorescent reagent allows for more sensitive analysis. Fluorescent probes for β-gal have the basic property of emitting fluorescence upon hydrolysis of β-glycosidic bond . They include fluorescein Di-β-D-galactopyranoside (FDG) and 9H-(1,3-Dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-galactopyranoside (DDAO galactoside). Appropriate fluorescent probes should be selected based on differences in fluorescence wavelengths and efficiency of introduction into the cells.

References

1. "How to select and use fluorescent and luminescent reagents for successful experiments” ed. by Miwa, Y. Yodosha, Japan, (2007). (Japanese).