| 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. Gene Cloning: In gene cloning, X-gal is used as a visual indication of whether a cell expresses a functional beta-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 alpha-complementation of the beta-galactosidase gene, where a fragment of the lacZ gene (lacZalpha) in the plasmid can complement another mutant lacZ gene (lacZdeltaM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZalpha is transformed into a lacZdeltaM15 cells, they form a functional beta-galactosidase. The presence of an active beta-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 lacZalpha gene. Successful ligation therefore disrupts the lacZalpha gene, alpha-complementation is therefore also disrupted and no functional beta-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 DH5alpha which carries the mutant lacZdeltaM15 gene. Protein-protein Interactions: In two-hybrid analysis, beta-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 beta-galactosidase, which results in the formation of blue-pigmented colonies in the presence of X-gal, will therefore indicate a successful interaction between proteins.[10] This technique may be limited to screening libraries of size of less than around 106.[10] The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of indole. Synonyms: X-Gal, 5-Bromo-4-chloro-3-indolyl-b-D-galactoside (galactopyranoside), BCIG CAS No: 7240-90-6 Molecular Formula: C14H15BrClNO6 Molecular Weight: 408.61 Purity (HPLC): 99% (HPLC), Single Spot (TLC) Appearance: White lyophilized powder Solubility: Soluble in DMF Water (KF): 1% Specific Rotation: -60 to -64 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 -20C. Stable for up to 12 months after receipt. Protect from light. Handling and Storage of X-Gal: X-Gal is stable at RT for short periods of time. For long term storage, store X-Gal in a sealed desiccated container at -20C. Do NOT expose to light. Warm the desiccator to RT for one hour prior to removing. X-Gal is hygroscopic and it is critical to avoid any condensation of water on the X-Gal crystals. X-Gal Stability: X-Gal in original powder form should be stable for at least two years, possibly longer unopened. X-Gal Solution is stable for 6-12 months at -20C. However, frequent use will degrade the solution over time and we recommend making new stock every 2-3 months depending on use. Also Available: X1000-05: XGAL, 40mg/ml in DMF |
