X-gal

X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3indolyl--D-galactopyranoside) is an organic compound

consisting of galactose linked to a substituted indole. The
compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964.[1] The formal chemical 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,
-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 enzymecatalyzed hydrolysis.[2]

zyme 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 (lacZM15)
in the cell. Both genes by themselves produce nonfunctional peptides, however, when expressed together,
as when a plasmid containing lacZ is transformed into a
lacZM15 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 identied 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 specic E. coli host strains
such as DH5 which carries the mutant lacZM15 gene.

Uses

X-gal is an analog of lactose, and therefore may be hydrolyzed by the -galactosidase enzyme which cleaves the
-glycosidic bond in D-lactose. X-gal, when cleaved by
-galactosidase, yields galactose and 5-bromo-4-chloro3-hydroxyindole. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloroindigo, an intensely blue product which is insoluble. Xgal itself is colorless, the presence of blue-colored product therefore may be used as a test for the presence
of an active -galactosidase. This easy identication
of an active enzyme allows the gene for -galactosidase 1.2.1 Variants
(the lacZ gene) to be used as a reporter gene in various
X-gal has a number of variants, which are similar
applications.[3]
molecules with slight dierences serving mainly to produce colors other than blue as a signal.

1.1

Reaction
1.3 Protein-protein interactions

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
1.2 Cloning
of X-gal, will therefore indicate a successful interaction
In gene cloning, X-gal is used as a visual indication of between proteins.[10] This technique may be limited to
whether a cell expresses a functional -galactosidase en- screening libraries of size of less than around 106 .[10] The
1

successful cleavage of X-gal also creates a noticeably foul

odor due to the volatilization of indole.

See also
X-Gluc

References

[1] Horwitz JP and 7 others, 1964. Substrates for cytochemical demonstration of enzyme activity. I. Some substituted 3-indolyl--D-glycopyranosides. Journal of Medicinal Chemistry 7: 574-575.
[2] Kiernan JA 2007. Indigogenic substrates for detection
and localization of enzymes. Biotechnic & Histochemistry 82(2): 73-103.
[3] Sandhu, Sardul Singh (2010). Recombinant DNA Technology. I K International Publishing House. p. 116. ISBN
978-9380578446.
[4] 5-Bromo-3-indolyl -D-galactopyranoside. Retrieved 4
February 2014.
[5] Salmon-Gal - PubChem. Retrieved 4 February 2014.
[6] Purple-beta-D-Gal - PubChem. Retrieved 4 February
2014.
[7] 5-Bromo-6-chloro-3-indolyl--D-glucopyranoside.
Retrieved 4 February 2014.
[8] Green--D-Gal - Biotium, Inc.. Retrieved 4 February
2014.
[9] 4-Methylumbelliferyl -D-galactopyranoside.
trieved 4 February 2014.

Re-

[10] Joung J, Ramm E, Pabo C (2000). A bacterial twohybrid selection system for studying protein-DNA and
protein-protein interactions. Proc Natl Acad Sci USA
97 (13): 73827. doi:10.1073/pnas.110149297. PMC
16554. PMID 10852947.

REFERENCES

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