X-Gal, Bluo-Gal, & Magenta-Gal

Bluo-Gal is used as an alternative to X-Gal, and produces a darker blue color. Bluo-Gal is hydrolyzed by β-galactosidase to produce a blue precipitate. This precipitate is insoluble in alcohol and xylenes, thus making it suitable for immunoblotting and immunocytochemical assays. Bacterial colonies containing active β-galactosidase produce blue colonies when grown on plates containing Bluo-Gal.

Magenta-Gal is used as an alternative to X-Gal, and produces a red color. Magenta-Gal is hydrolyzed by β-galactosidase to produce a red precipitate. This precipitate is insoluble in alcohol and xylenes, thus making it suitable for immunoblotting and immunocytochemical assays. Bacterial colonies containing active β-galactosidase produce red colonies when grown on plates containing Magenta-Gal.

Magenta-Gal is commonly used in conjunction with IPTG (GoldBIo #I2481) for red/white screening.

5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside is most commonly known as X-Gal. X-gal is a histochemical substrate for β-galactosidase which cleaves X-Gal to yield an insoluble blue precipitate.

X-Gal is a mainstay in molecular biology cloning applications in which it is used to detect the activity of β-galactosidase. X-Gal is used to detect the insertion of foreign DNA into the lacZ region of a plasmid DNA. Insertion of DNA into the lacZ region results in the loss of β-galactosidase activity. Bacteria cells that retain active β-galactosidase will result in characteristic blue colonies. Successful disruption of the lacZα gene disrupts the α-complementation of the β-galactosidase gene and the precipitate does not form, resulting in white colonies.

Many other applications also use X-Gal as a substrate to detect β-galactosidase activity. These include β-galactosidase -antibody linked immunoassays and immunohistochemistry, coliphage detection based on β-galactosidase induction and the detection of micrometastasis formation during tumor progession.