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.
|β-Galactosidase Detection Reagents Selection Guide|
|Wavelength (ex/em)||493,525/560||490/514||497/518||615 nm||420 nm|
|For Live Cell||○||×||△||×||×|
|For Fixed Cell||○||△||△||○||△|
|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|
* This Conc. is for ELISA format.