X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) is widely used as a chromogenic substrate for β-Galactosidase in the reporter gene assay. X-Gal is a small molecule which consists of Galactose moiety and 5-bromo-4-chloro-3-indole moiety. When X-Gal is cleaved, an insoluble blue precipitate is formed.

Fluorescein Di-β-D-galactopyranoside (FDG), which is a fluorescein-based galactosidase substrates, and is used for detection of β-Galactosidase. This substrate forms a soluble green fluorescent product upon hydrolysis.

Although FDG enters viable cells, its permeability is insufficient for the use of live cell imaging. C12FDG, which is a variant of FDG, has been modified to have higher cell permeability. C12FDG can be loaded in the live cells; however, high concentration and long incubation time are required for loading into live cells.

SPiDER-βGal is developed to overcome these issues. By the enzymatic reaction, SPiDER-βGal produces a quinone methide and forms covalent bond with nucleophic functional groups of the proteins in the vicinity of the molecules, producing a fluorescent product that is well retained by cells. Unlike commercially available substrates, SPiDER-βGal allows a single-cell analysis under the lowest level of substrate concentration.

Beta-galactosidase Detection

β-Galactosidase Detection Reagents Selection Guide
Product Name SPiDER-βGal FDG C12FDG X-Gal ONPG
Measurement Fluorescence Fluorescence Fluorescence Colorimetric Colorimetric
Wavelength (ex/em) 493,525/560 490/514 497/518 615 nm 420 nm
Detection Method Fluorescence
Flow cytometry
Flow cytometry
Flow cytometry
Microscope Absorbance
microplate reader
For Live Cell × × ×
For Fixed Cell
Intracellular Retention × ×
Final Conc. of Cell Staining 1 µmol/l 100 ~ 2 mmol/l 33 µmol/l 2.5 mmol/l 2.3 mmol/l~*
Incubation Time of Cell Staining 15 min. 20-25 min. 20-60 min. Overnight N/A
Number of Enzyme Reactive Site 1 2 2 1 1
Product Code SG02-10 - - - -

* This Conc. is for ELISA format.

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