X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an
organic compound
Some chemical authorities define an organic compound as a chemical compound that contains a carbon–hydrogen or carbon–carbon bond; others consider an organic compound to be any chemical compound that contains carbon. For example, carbon-co ...
consisting of
galactose
Galactose (, ''wikt:galacto-, galacto-'' + ''wikt:-ose#Suffix 2, -ose'', ), sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweetness, sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epime ...
linked to a substituted
indole
Indole is an organic compound with the formula . Indole is classified as an aromatic heterocycle. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indoles are derivatives of indole ...
. The compound was synthesized by
Jerome Horwitz and collaborators in 1964. 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 often used in
molecular biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
to test for the presence of an enzyme,
β-galactosidase
β-Galactosidase (EC 3.2.1.23, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase) is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. (This enzym ...
, in the place of its usual target, a β-galactoside. It is also used to detect activity of this enzyme in
histochemistry
Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Albert Hewett ...
and
bacteriology
Bacteriology is the branch and specialty of biology that studies the Morphology (biology), morphology, ecology, genetics and biochemistry of bacteria as well as many other aspects related to them. This subdivision of microbiology involves the iden ...
. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to
indigo dye
Indigo dye is an organic compound with a distinctive indigo, blue color. Indigo is a natural dye obtained from the leaves of some plants of the Indigofera#Uses, ''Indigofera'' genus, in particular ''Indigofera tinctoria''. Dye-bearing ''Indigofer ...
as a result of enzyme-catalyzed hydrolysis.
A less often used but very similar (
chiral
Chirality () is a property of asymmetry important in several branches of science. The word ''chirality'' is derived from the Greek language, Greek (''kheir''), "hand", a familiar chiral object.
An object or a system is ''chiral'' if it is dist ...
) compound is X-
-gal (Xαgal, X-alpha-gal), or 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, which is hydrolyzed by
α-galactosidase (
EC 3.2.1.22) instead of β-galactosidase (EC 3.2.1.23).
Uses
X-gal is an
analog of
lactose
Lactose is a disaccharide composed of galactose and glucose and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from (Genitive case, gen. ), the Latin word for milk, plus the suffix ''-o ...
, and therefore may be hydrolyzed by the
β-galactosidase
β-Galactosidase (EC 3.2.1.23, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase) is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. (This enzym ...
enzyme which cleaves the β-
glycosidic bond
A glycosidic bond or glycosidic linkage is a type of ether bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate.
A glycosidic bond is formed between the hemiacetal or hemiketal group o ...
in -lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole - 1. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-
indigo
InterGlobe Aviation Limited (d/b/a IndiGo), is an India, Indian airline headquartered in Gurgaon, Haryana, India. It is the largest List of airlines of India, airline in India by passengers carried and fleet size, with a 64.1% domestic market ...
- 2, an intensely blue product which is insoluble. X-gal itself is colorless, so the presence of blue-colored product may therefore be used as a test for the presence of active β-galactosidase. This also allows for bacterial β-galactosidase (so called ''lacZ '') to be used as a
reporter
A journalist is a person who gathers information in the form of text, audio or pictures, processes it into a newsworthy form and disseminates it to the public. This is called journalism.
Roles
Journalists can work in broadcast, print, advertis ...
in various applications.
Similarly, Xαgal is used as a reporter compound for
α-galactosidase (e.g. Mel1 in yeast).
Reaction
Cloning
In
gene cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
, X-gal is used as a visual indication of whether a cell expresses a functional
β-galactosidase
β-Galactosidase (EC 3.2.1.23, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase) is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. (This enzym ...
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 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 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 ''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. Often, the plate containing X-Gal also contains IPTG (isopropyl β--1-thiogalactopyranoside). IPTG is a chemical structure analogue of lactose. However, IPTG cannot be hydrolyzed by β-galactosidase. IPTG is used as an inducer that binds to lac repressor releasing the DNA and allowing transcription. The presence of IPTG in the agar plate therefore increases the synthesis of β-galactosidase.
Variants
X-gal has a number of variants, which are similar molecules with slight differences serving mainly to produce colors other than blue as a signal.
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 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 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.
[{{cite journal
, vauthors=Joung J, Ramm E, Pabo C
, title=A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions
, journal=Proc Natl Acad Sci USA
, volume=97
, issue=13
, pages=7382–7
, year=2000
, pmid=10852947
, doi=10.1073/pnas.110149297
, pmc=16554
, bibcode=2000PNAS...97.7382J
, doi-access=free
] This technique may be limited to screening libraries of size of less than around 10
6.
The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of
indole
Indole is an organic compound with the formula . Indole is classified as an aromatic heterocycle. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indoles are derivatives of indole ...
.
See also
*
X-Gluc
References
Galactosides
Indoles
Chloroarenes
Bromoarenes