X-Gal (5-Bromo-4-chloro-3-indolyl-b-D-galactoside galactopyranoside, Xgal)
|Biochemicals||Storage: -20°CShipping: Blue Ice|
Blue substrate used in the detection of b-galactosidase in bacteria or phage as a selection agent for cloning experiments utilizing the lacZ vector. Colonies expressing b-galactosidase will appear blue in the presence of XGAL. Others will appear white.
In gene cloning, X-gal is used as a visual indication of whether a cell expresses a functional -galactosidase enzyme in a technique called blue/white screening. This method of screening is a convenient way of distinguishing a successful cloning product from other unsuccessful ones.
The blue/white screening method relies on the principle of -complementation of the -galactosidase gene, where a fragment of the lacZ gene (lacZ ) in the plasmid can complement another mutant lacZ gene (lacZ M15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ is transformed into a lacZ M15 cells, they form a functional -galactosidase. The presence of an active -galactosidase may be detected when cells are grown in plates containing X-gal, the blue-colored product precipitated within cells resulted in the characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZ gene. Successful ligation therefore disrupts the lacZ gene, -complementation is therefore also disrupted and no functional -galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones. Example of cloning vectors used for this test are pUC19, pBluescript, pGem-T Vectors, and it also requires the use of specific E. coli host strains such as DH5 which carries the mutant lacZ M15 gene.
In two-hybrid analysis, -galactosidase may be used as a reporter to identify proteins that interact with each other. In this method, genome libraries may be screened for protein interaction using yeast or bacterial system. Where there is a successful interaction between proteins being screened, it will result to the binding of an activation domain to a promoter. If the promoter is linked to a lacZ gene, the production of -galactosidase, which results in the formation of blue-pigmented colonies in the presence of X-gal, will therefore indicate a successful interaction between proteins. This technique may be limited to screening libraries of size of less than around 106. The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of indole.
Purity (HPLC): 99%
Purity (TLC): Single Spot
Appearance: White lyophilized powder
Solubility: Soluble in DMF
Water (KF): 1%
Specific Rotation: -60º to -64º
Identity by I.R. or NMR: Conforms to structure
RNase, DNase or Proteases: None Detected
Quality Control (Blue-White Assay):
[LacZ+]: Pale blue center/dense blue periphery
[LacZ-]: Faint blue center/white periphery
Storage and Stability:
Store at -20°C. Stable for 6 months after receipt. Protect from light.
X1000-05: XGAL, 40mg/ml in DMF
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.