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.
X-gal is commonly used in conjunction with IPTG (GoldBio #I2481) for blue-white screening.
MOLECULAR BIOLOGY GRADE
MW: 408.63 g/mol
Purity: Greater than 99% (HPLC)
Solution: Clear (2% in DMF)
Biological Activity: Blue color with Lambda, pUC, and M13
Storage/Handling: Store at -20°C. Protect from Light.
PubChem Chemical ID:
X-Gal Quick Answers
X-gal is the chromogenic substrate for the enzyme β-galactosidase. When X-gal is cleaved by
β-galactosidase, it forms galactose and 5-bromo-4-chloro-3-hydroxyindole.
5-bromo-4-chloro-3-hydroxyindole can dimerize and then is oxidized, forming 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product.
The blue product suggests the
presence of β-galactosidase. Therefore, this substrate is
often used as a reporter in molecular biology.
Instructions to prepare a 20 mg/ml X-gal stock solution are as follows
1. Weigh 200 mg of X-Gal into a 15 ml polypropylene centrifuge tube.
2. Add 10 mL of Dimethylformamide (DMF). Vortex to dissolve the X-Gal.
3. Store at -20°C. Wrap the centrifuge tube in aluminum foil to protect from light.
Note: The X-Gal solution is stable for 6-12 months unless improperly handled. We recommend making 1 ml aliquot portions of the solution to minimize degradation if there will be frequent small scale use of the X-Gal solution.
For blue-white screening, use 10 µl per 1 mL of media if you’re using a 20 mg/ml stock solution. If you’re using a 100 mg/ml stock solution, which is only stable for 1 week, use 2 µl.
- Store powder X-gal at -20°C. Protect from Light.
- Store X-gal stock solution at -20°C. It’s important to protect your stored stock solution from light. You can do this by wrapping the tube in aluminum foil.
|Grade||MOLECULAR BIOLOGY GRADE|
|Storage/Handling||Store at -20°C. Protect from light.|
Protocol for adding X-Gal and IPTG to agar or plates for Blue/White screening method of bacterial transformation
Procedure for noncovalent protein internalization using β-Gal and X-Gal, by Wang et al. (2006).
Sample Certificate of Analysis - X-gal
COA for example only. Actual tests may differ.