beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.
beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use.
Gong
Haibiao
H
LI-COR Biosciences, Lincoln, NE 68504, USA. herbert.gong@licor.com
Zhang
Bin
B
Little
Garrick
G
Kovar
Joy
J
Chen
Huaxian
H
Xie
Wen
W
Schutz-Geschwender
Amy
A
Olive
D Michael
DM
D000166:Acridines / Q000032:analysis; D000818:Animals; D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D008722:Methods; D012107:Research Design; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
C416725:9H-(1,3-dichloro-9,9-dimethylacridine-2-on-7-yl)phosphate; D000166:Acridines; D001616:beta-Galactosidase