5-Bromo-4-chloro-3-indolyl-beta-D-galactoside

5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷

X-Gal is a cell-permeable, chromogenic substrate for β-galactosidase that is used in histochemical and molecular biology applications, including detection of lacZ activity in cells and tissues. It generates an indigo-colored precipitate as an indication of enzymatic activity.

5-bromo-4-chloro-3-indolyl beta-D-galactoside is an indolyl carbohydrate that is the beta-D-galactoside of 3-hydroxy-1H-indole in which the indole moiety is substituted at positions 4 and 5 by chlorine and bromine, respectively. It is used to test for the presence of an enzyme, beta-galactosidase, which cleaved the glycosidic bond to give 5-bromo-4-chloro-3-hydroxy-1H-indole, which immediately dimerises to give an intensely blue product. It has a role as a chromogenic compound. It is an indolyl carbohydrate, a beta-D-galactoside, an organochlorine compound, an organobromine compound and a D-aldohexose derivative.

X-Gal is a chromogenic substrate for β-galactosidase, yielding a blue precipitate. X-Gal is an indicator for coliforms in culture media, as well as for the detection of coliforms (E. coli ) in municipal water supplies and food products. X-Gal can be used in conjuction with the inducer IPTG, which binds and inhibits the lac repressor thus inducing β-galactosidase expression. X-Gal is the substrate of choice for blue-white selection of recombinant bacterial colonies with the lac+ genotype.

Upon hydrolysis, X-Gal yields a localized, insoluble, blue precipitate, making it exceptionally useful in blotting, immunocytochemical, and ELISA assays. 5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-β-D-galactoside, X-Gal) is a chromogenic indicator for β-galactosidase activity. The colorless product is cleaved by lactose-utilizing bacteriain liquid media or on agar plates and yields a blue-green colored precipiate.

X-Gal can also be used as a detecting agent for β-galactosidase activity in reporter gene assays or for identification of positive transformants on agar plates when using the α-complementation approach. Color formation is due to dimerization and oxidation of 5-bromo-4-chloro-3-hydroxyindole which results from enzymatic cleavage of X-Gal. The reaction proceeds only in the presence of oxygen, i.e. in aerobiccultures. Well detectable color bacterial colonies or liquid cultures is obtained within 16-48 h. In the presence of purified β-galactosidase, color develops within minutes to few hours.

Title: X-gal

CAS Registry Number: 7240-90-6

CAS Name: 5-Bromo-4-chloro-1H-indol-3-yl b-D-galactopyranoside

Molecular Formula: C14H15BrClNO6

Molecular Weight: 408.63

Percent Composition: C 41.15%, H 3.70%, Br 19.55%, Cl 8.68%, N 3.43%, O 23.49%

Literature References: Chromogenic substrate of the lacZ gene product, b-galactosidase; produces a rich blue color when cleaved by the enzyme. Prepn: J. P. Horwitz et al., J. Med. Chem. 7, 574 (1964). Colorimetric reaction in tissue to localize b-galactosidase: B. Pearson et al., Lab. Invest. 12, 1249 (1963); Z. Lojda, Histochemie 23, 266 (1970). Protocol for blue-white a-complementation screening: J. Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York, 2nd ed., 1989) pp 1.7-1.9, 1.85-1.87. Optimization of detection of exogenous bacterial lacZ expression: D. J. Weiss et al., Hum. Gene Ther. 8, 1545 (1997); of fixative conditions in X-gal staining: W. Ma et al., J. Histochem. Cytochem. 50, 1421 (2002); of blue-white screening procedure: A. L. Sherwood, BioTechniques 34, 644 (2003).

Properties: Amorphous solid from methanol, mp 237-239° (dec). [a]D24 -69° (c = 1.0 in 50% DMF). Sol in DMF, DMSO.

Melting point: mp 237-239° (dec)

Optical Rotation: [a]D24 -69° (c = 1.0 in 50% DMF)

Use: In histochemical staining, in blue-white selection of bacterial colonies with lacZ gene activity during cloning experiments.

COA:

Name: 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside, X-gal   

 CAS: 7240-90-6    M.F.: C₁₄H₁₅BrClNO₆      M.W.: 408.63   

References:

1. Weiss DJ, Liggitt D, Clark JG, Human Gene Therapy 1997, 8, 13, p1545-1554
2. Engler KH, et al., J. Microbiol. Methods 1999, 35, p207
3. Arakawa H, et al., Anal. Biochem. 1991, 199, p238