A fluorescent substrate for β-galactosidase
Fluorescein di-β-galactopyranoside is a fluorogenic substrate for β-galactosidase.1 Upon enzymatic cleavage by β-galactosidase, fluorescein is released and its fluorescence can be used to quantify β-galactosidase activity. Fluorescein displays excitation/emission maxima of 490/514 nm, respectively.2,1
1.Plovins, A., Alvarez, A.M., Iba?ez, M., et al.Use of fluorescein-di-β-D-galactopyranoside (FDG) and C12-FDG as substrates for β-galactosidase detection by flow cytometry in animal, bacterial, and yeast cellsAppl. Environ. Microbiol.60(12)4638-4641(1994) 2.Hermanson, G.T.Fluorescent ProbesBioconjugate Techniques(2013)
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Cell experiment: |
The cells (5×103 cells per well) are cultured in a 96-well plate overnight for attachment, washed, and then fixed in solutions. An aliquot (100 μL) of the reaction buffer (i.e., the staining solution without X-Gal) is added into each well. Then, 10 μL of 2 mM Fluorescein di(β-D-galactopyranoside) is added per well and the plate is incubated in the dark at 37°C for 24 h without CO2 supply. After incubation at 37°C for 24 h, 100 μL of the supernatant is transferred to a 96-well plate for fluorescent measurement in triplicates. The fluorescein fluorescence is measured using a fluorometer with an excitation at 485 nm and an emission at 535 nm[1]. |
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参考文献: [1]. Yang NC, et al. A fluorimetric method using fluorescein di-beta-D-galactopyranoside for quantifying the senescence-associated beta-galactosidase activity in human foreskin fibroblast Hs68 cells. Anal Biochem. 2004 Feb 15;325(2):337-43. |
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