X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) is widely used as a chromogenic substrate for ß-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-ß-D-galactopyranoside (FDG), which is a fluorescein-based galactosidase substrates, and is used for detection of ß-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.
To overcome these issues, Dojindo has introduced SPiDER-ßGal. By the enzymatic reaction, SPiDER-ßGal 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-ßGal allows a single-cell analysis under the lowest level of substrate concentration.
ß-Galactosidase Detection Reagents Selection Guide |
|||||
|---|---|---|---|---|---|
| Product Name | SPiDER-ßGal Read More |
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 microscope Flow cytometry |
Fluorescence microscope Flow cytometry |
Fluorescence microscope Flow cytometry |
Microscope | Absorbance microplate reader |
| For Live Cell | ○ | × | △ | × | × |
| For Fixed Cell | ○ | △ | △ | ○ | △ |
| Intracelluler 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.
