Although chromogenic assays of beta-galactosidase activity (i.e. X-Gal) are still widely used, the recent advance in fluorescence instrumentation has made fluorogenic substrates (such as FDG and MUG) be several orders of magnitude more sensitive. FDG is considered to be one of the most sensitive fluorogenic substrates available for detecting beta-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 ?-galactosidase in a chromogenic mode since the enzymatic product (fluorescein) exhibits a large extinction coefficient (close to 100,000 cm-1mol-1). FDG has been widely used for identifying LacZ-positive cells with fluorescence microscopy and flow cytometry. It is also used to detect beta-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.
京公网安备11010802025653 版权所有：北京逸优科技有限公司
