β-galactosidase Detection - Amerigo Scientific

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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 microscope Flow cytometry	Fluorescence microscope Flow cytometry	Fluorescence microscope 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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