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| ImageFile = X-Gal.svg
| ImageSize = 200px
| IUPACName = 5-
| SystematicName = (2''S'',3''R'',4''S'',5''R'',6''R'')-2-[(5-Bromo-4-chloro-1''H''-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol
| OtherNames =
| Section1 = {{Chembox Identifiers
| CASNo_Ref = {{cascite|correct|??}}
| CASNo = 7240-90-6
| UNII_Ref = {{fdacite|correct|FDA}}
| UNII = V595OG374W
| PubChem = 65181
| ChemSpiderID_Ref = {{chemspidercite|changed|chemspider}}
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'''X-gal''' (also abbreviated '''BCIG''' for 5-bromo-4-chloro-3-indolyl-β-{{small|D}}-galactopyranoside) is an [[organic compound]] consisting of [[galactose]] linked to a substituted [[indole]]. The compound was synthesized by [[Jerome Horwitz]] and collaborators
A less often used but very similar ([[Chirality|chiral]]) compound is '''X-<math>\alpha</math>-gal''' (Xαgal, X-alpha-gal), or 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, which is hydrolyzed by [[Α-Galactosidase|α-galactosidase]] ([[Enzyme Commission number|EC]] 3.2.1.22) instead of β-galactosidase (EC 3.2.1.23).<ref>{{Cite web |title=Information on EC 3.2.1.22 - alpha-galactosidase - BRENDA Enzyme Database |url=https://www.brenda-enzymes.org/enzyme.php?ecno=3.2.1.22 |access-date=2023-07-31 |website=www.brenda-enzymes.org}}</ref><ref>{{Cite web |title=Information on EC 3.2.1.23 - beta-galactosidase - BRENDA Enzyme Database |url=https://www.brenda-enzymes.org/enzyme.php?ecno=3.2.1.23 |access-date=2023-07-31 |website=www.brenda-enzymes.org}}</ref>
==Uses==
X-gal is an [[Structural analog|analog]] of [[lactose]], and therefore may be hydrolyzed by the [[beta-galactosidase|β-galactosidase]] enzyme which cleaves the β-[[glycosidic bond]] in {{small|D}}-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 dye|indigo]] - '''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.
Similarly, Xαgal is used as a reporter compound for [[Α-Galactosidase|α-galactosidase]] (e.g. Mel1 in yeast).<ref>{{Cite journal |last1=Goddard |first1=Alan |last2=Ladds |first2=Graham |last3=Davey |first3=John |date=2005-01-15 |title=Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2 |url=https://pubmed.ncbi.nlm.nih.gov/15580593 |journal=Yeast (Chichester, England) |volume=22 |issue=1 |pages=31–41 |doi=10.1002/yea.1190 |issn=0749-503X |pmid=15580593|s2cid=33926866 }}</ref>
===Reaction===
[[Image:X-Gal reaction.png|550px]]
===Cloning ===
In [[Clone (genetics)|gene cloning]], X-gal is used as a visual indication of whether a cell expresses a functional [[beta-galactosidase|β-galactosidase]] enzyme in a technique called [[Blue white screen|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
====Variants====
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! Short name !! Long name !! Color
|-
| Blue-Gal, Bluo-Gal || 5-Bromo-3-indolyl β-{{small|D}}-galactopyranoside || Dark blue<ref>{{cite web|url=http://www.sigmaaldrich.com/catalog/product/sigma/b4387?lang=en®ion=US |title= 5-Bromo-3-indolyl β-D-galactopyranoside |
|-
| Rose-Gal, Salmon-Gal, Y-Gal, Red-Gal || 6-Chloro-3-indolyl-β-{{small|D}}-galactopyranoside || Pink<ref>{{cite web|url=https://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=24854972 |title=Salmon-Gal - PubChem |
|-
| Purple-β-{{small|D}}-Gal || 5-Iodo-3-indolyl-β-{{small|D}}-galactopyranoside || Purple<ref>{{cite web|url=https://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=24896110|title=Purple-beta-D-Gal - PubChem |
|-
| Magenta glucoside, Magenta-GLC, Magenta gal || 5-Bromo-6-chloro-3-indolyl-β-{{small|D}}-glucopyranoside || Magenta<ref>{{cite web|url=http://www.sigmaaldrich.com/catalog/product/sigma/74781?lang=en®ion=US |title=5-Bromo-6-chloro-3-indolyl-β-D-glucopyranoside |
|-
| Green-β-{{small|D}}-
|-
| MUG, MUGA || 4-Methylumbelliferyl β-{{small|D}}-galactopyranoside || Fluorescent<ref>{{cite web|url=http://www.sigmaaldrich.com/catalog/product/sigma/m1633?lang=en®ion=US |title=4-Methylumbelliferyl β-D-galactopyranoside |
|}
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|year=2000
|pmid=10852947
|doi=10.1073/pnas.110149297
|pmc=16554
|bibcode=2000PNAS...97.7382J
|doi-access=free
}}</ref> This technique may be limited to screening libraries of size of less than around 10<sup>6</sup>.<ref name="joung"/> The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of [[indole]].
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