5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-Gal)
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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
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X-GalX-Gal is the normal abbreviation for 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside; less commonly the abbreviation BCIG is also used.
X-Gal is enzymatically hydrolysed by β-galactosidase to yield galactose and 5-bromo-4-chloro-3-hydroxyindole. The soluble, colourless, indole hydrolysis product is subsequently transformed into an insoluble blue compound, 5,5’-dibromo-4,4’-dichloro indigo, via a dimerisation and oxidation sequence.
X-Gal is frequently used in molecular biology applications to indicate the activity of β-galactosidase, an enzyme encoded by the lacZ gene. In gene cloning, a X-Gal blue/white colony screening assay can be used to differentiate recombinant colonies which stain white from non-recombinant ones which stain blue.1 The lacZ gene is a classical reporter gene, in yeast two-hybrid analysis allowing a distinction to be made between bacteria where a successful interaction resulting in binding of an activation domain to a promoter is made and bacteria in which such an interaction is not made. Linkage of the promoter to the lacZ gene will result in the production of the blue colour of 5,5’-dibromo-4,4’-dichloro indigo (by production of β-galactosidase and the subsequent hydrolysis, dimerisation and oxidation of X-Gal) by colonies hosting a successful interaction.2
1. J Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, 3rd
Edition, Cold Spring Harbor Laboratory Press, 2001.
2. JK Joung et al, Proc. Natl. Acad. Sci. USA 2000, 97, 7382