Fluorescein di-β-D-galactopyranoside (FDG) is one of the most sensitive fluorogenic substrates available for detecting β-galactosidase. The colorless and nonfluorescent FDG is hydrolyzed to highly fluorescent fluorescein, which exhibits excellent spectral properties (Ex/Em=492/520 nm) that match the optimal detection window of most fluorescence instruments. Galactosidase-catalyzed hydrolysis of FDG can be followed by fluorescence increase around 520 nm. Alternatively, FDG can also be used to detect β-galactosidase in a chromogenic mode since the enzymatic product (fluorescein) exhibits a large extinction coefficient (close to 100,000 cm-1mol-1). In general, fluorescence-based measurements can be several orders of magnitude more sensitive than absorption-based measurements. FDG has been widely used for identifying lacZ-positive cells with fluorescence microscopy and flow cytometry. FDG is also used to detect β-galactosidase expression in live cells. Fluorescence-based assays employing FDG are also reported to be 100 to 1000-fold more sensitive than radioisotope-based ELISAs.
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