X-gal
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X-gal X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and col- laborators in Detroit, MI, in 1964. [1] The formal chem- ical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl may be the source of the X in the X-gal contraction. X-gal is much used in molecular biology to test for the presence of an enzyme, β-galactosidase. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo as a result of enzyme- catalyzed hydrolysis. [2] 1 Uses X-gal is an analog of lactose, and therefore may be hy- drolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro- 3-hydroxyindole. The latter then spontaneously dimer- izes and is oxidized into 5,5'-dibromo-4,4'-dichloro- indigo, an intensely blue product which is insoluble. X- gal itself is colorless, the presence of blue-colored prod- uct therefore may be used as a test for the presence of an active β-galactosidase. This easy identification of an active enzyme allows the gene for β-galactosidase (the lacZ gene) to be used as a reporter gene in various applications. [3] 1.1 Reaction 1.2 Cloning In gene cloning, X-gal is used as a visual indication of whether a cell expresses a functional β-galactosidase en- zyme in a technique called blue/white screening. This method of screening is a convenient way of distinguish- ing a successful cloning product from other unsuccessful ones. The blue/white screening method relies on the princi- ple of α-complementation of the β-galactosidase gene, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non- functional peptides, however, when expressed together, as when a plasmid containing lacZα is transformed into a lacZΔM15 cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be de- tected when cells are grown in plates containing X-gal, the blue-colored product precipitated within cells resulted in the characteristic blue colonies. However, the mul- tiple cloning site, where a gene of interest may be lig- ated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα gene, α-complementation is therefore also disrupted and no functional β-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 DH5α which carries the mutant lacZΔM15 gene. 1.2.1 Variants X-gal has a number of variants, which are similar molecules with slight differences serving mainly to pro- duce colors other than blue as a signal. 1.3 Protein-protein interactions In two-hybrid analysis, β-galactosidase may be used as a reporter to identify proteins that interact with each other. In this method, genome libraries may be screened for pro- tein 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 do- main to a promoter. If the promoter is linked to a lacZ gene, the production of β-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 10 6 . [10] The 1
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Transcript of X-gal
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X-gal
X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl--D-galactopyranoside) is an organic compoundconsisting of galactose linked to a substituted indole. Thecompound was synthesized by Jerome Horwitz and col-laborators in Detroit, MI, in 1964.[1] The formal chem-ical name is often shortened to less accurate but alsoless cumbersome phrases such as bromochloroindoxylgalactoside. The X from indoxyl may be the source ofthe X in the X-gal contraction. X-gal is much used inmolecular biology to test for the presence of an enzyme,-galactosidase. It is also used to detect activity of thisenzyme in histochemistry and bacteriology. X-gal is oneof many indoxyl glycosides and esters that yield insolubleblue compounds similar to indigo as a result of enzyme-catalyzed hydrolysis.[2]
1 UsesX-gal is an analog of lactose, and therefore may be hy-drolyzed by the -galactosidase enzymewhich cleaves the-glycosidic bond in D-lactose. X-gal, when cleaved by-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter then spontaneously dimer-izes and is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an intensely blue product which is insoluble. X-gal itself is colorless, the presence of blue-colored prod-uct therefore may be used as a test for the presenceof an active -galactosidase. This easy identicationof an active enzyme allows the gene for -galactosidase(the lacZ gene) to be used as a reporter gene in variousapplications.[3]
1.1 Reaction
1.2 CloningIn gene cloning, X-gal is used as a visual indication ofwhether a cell expresses a functional -galactosidase en-
zyme in a technique called blue/white screening. Thismethod of screening is a convenient way of distinguish-ing a successful cloning product from other unsuccessfulones.The blue/white screening method relies on the princi-ple of -complementation of the -galactosidase gene,where a fragment of the lacZ gene (lacZ) in the plasmidcan complement another mutant lacZ gene (lacZM15)in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together,as when a plasmid containing lacZ is transformed into alacZM15 cells, they form a functional -galactosidase.The presence of an active -galactosidase may be de-tected when cells are grown in plates containing X-gal,the blue-colored product precipitated within cells resultedin the characteristic blue colonies. However, the mul-tiple cloning site, where a gene of interest may be lig-ated into the plasmid vector, is located within the lacZgene. Successful ligation therefore disrupts the lacZgene, -complementation is therefore also disrupted andno functional -galactosidase can form, resulting in whitecolonies. Cells containing successfully ligated insert canthen be easily identied by its white coloration from theunsuccessful blue ones. Example of cloning vectors usedfor this test are pUC19, pBluescript, pGem-T Vectors,and it also requires the use of specic E. coli host strainssuch as DH5 which carries the mutant lacZM15 gene.
1.2.1 Variants
X-gal has a number of variants, which are similarmolecules with slight dierences serving mainly to pro-duce colors other than blue as a signal.
1.3 Protein-protein interactions
In two-hybrid analysis, -galactosidase may be used as areporter to identify proteins that interact with each other.In this method, genome libraries may be screened for pro-tein interaction using yeast or bacterial system. Wherethere is a successful interaction between proteins beingscreened, it will result to the binding of an activation do-main to a promoter. If the promoter is linked to a lacZgene, the production of -galactosidase, which results inthe formation of blue-pigmented colonies in the presenceof X-gal, will therefore indicate a successful interactionbetween proteins.[10] This technique may be limited toscreening libraries of size of less than around 106.[10] The
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successful cleavage of X-gal also creates a noticeably foulodor due to the volatilization of indole.
2 See also X-Gluc
3 References[1] Horwitz JP and 7 others, 1964. Substrates for cytochem-
ical demonstration of enzyme activity. I. Some substi-tuted 3-indolyl--D-glycopyranosides. Journal of Medic-inal Chemistry 7: 574-575.
[2] Kiernan JA 2007. Indigogenic substrates for detectionand localization of enzymes. Biotechnic & Histochem-istry 82(2): 73-103.
[3] Sandhu, Sardul Singh (2010). Recombinant DNA Tech-nology. I K International Publishing House. p. 116. ISBN978-9380578446.
[4] 5-Bromo-3-indolyl -D-galactopyranoside. Retrieved 4February 2014.
[5] Salmon-Gal - PubChem. Retrieved 4 February 2014.
[6] Purple-beta-D-Gal - PubChem. Retrieved 4 February2014.
[7] 5-Bromo-6-chloro-3-indolyl--D-glucopyranoside.Retrieved 4 February 2014.
[8] Green--D-Gal - Biotium, Inc.. Retrieved 4 February2014.
[9] 4-Methylumbelliferyl -D-galactopyranoside. Re-trieved 4 February 2014.
[10] Joung J, Ramm E, Pabo C (2000). A bacterial two-hybrid selection system for studying protein-DNA andprotein-protein interactions. Proc Natl Acad Sci USA97 (13): 73827. doi:10.1073/pnas.110149297. PMC16554. PMID 10852947.
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