X-gal: Difference between revisions

| Verifiedfields = changed

| Watchedfields = changed

| verifiedrevid = 443011339

| IUPACName = 5-Bromo-4-chloro-1''H''-indol-3-yl β-{{small|D}}-galactopyranoside

| 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

| CASNo_Ref = {{cascite|correct|??}}

| CASNo = 7240-90-6

| UNII_Ref = {{fdacite|correct|FDA}}

| UNII = V595OG374W

| PubChem = 65181

| ChemSpiderID_Ref = {{chemspidercite|changed|chemspider}}

| ChemSpiderID = 58680

| ChEBI_Ref = {{ebicite|changed|EBI}}

| ChEBI = 75055

| SMILES = Brc3ccc2c(c(O[C@@H]1O[C@@H]([C@H](O)[C@H](O)[C@H]1O)CO)c[nH]2)c3Cl

| InChI = 1/C14H15BrClNO6/c15-5-1-2-6-9(10(5)16)7(3-17-6)22-14-13(21)12(20)11(19)8(4-18)23-14/h1-3,8,11-14,17-21H,4H2/t8-,11+,12+,13-,14-/m1/s1

| InChIKey = OPIFSICVWOWJMJ-AEOCFKNEBZ

| StdInChI_Ref = {{stdinchicite|changed|chemspider}}

| StdInChI = 1S/C14H15BrClNO6/c15-5-1-2-6-9(10(5)16)7(3-17-6)22-14-13(21)12(20)11(19)8(4-18)23-14/h1-3,8,11-14,17-21H,4H2/t8-,11+,12+,13-,14-/m1/s1

| StdInChIKey_Ref = {{stdinchicite|changed|chemspider}}

| StdInChIKey = OPIFSICVWOWJMJ-AEOCFKNESA-N

| MeSHName = X-gal}}

| Formula = C₁₄H₁₅BrClNO₆

| MolarMass = 408.629

| Appearance =

| Density =

| MeltingPt =

| BoilingPt =

| MainHazards =

| FlashPt =

| AutoignitionPt =

'''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 in 1964.<ref>Horwitz JP et al., 1964. Substrates for cytochemical demonstration of enzyme activity. I. Some substituted 3-indolyl-β-{{small|D}}-glycopyranosides. Journal of Medicinal Chemistry 7: 574-575.</ref> 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]] to test for the presence of an enzyme, [[beta-galactosidase|β-galactosidase]], in the place of its usual target, a β-galactoside. 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 dye]] as a result of enzyme-catalyzed hydrolysis.<ref>Kiernan JA 2007. Indigogenic substrates for detection and localization of enzymes. Biotechnic & Histochemistry 82(2): 73-103.</ref>

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>

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. This also allows for bacterial β-galactosidase (so called ''lacZ '') to be used as a [[reporter gene|reporter]] in various applications.<ref>{{cite book|title=Recombinant DNA Technology |first=Sardul Singh |last=Sandhu |year=2010 |publisher=I K International Publishing House |isbn=978-9380578446 |page=116}}</ref>

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===

===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 contains IPTG (isopropyl β-{{small|D}}-1-thiogalactopyranoside). IPTG is a chemical structure analogue of lactose.<ref>{{Cite web|url=https://www.bioline.com/iptg.html|title=IPTG - Bioline|website=www.bioline.com|language=en|access-date=2018-05-15}}</ref> 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.<ref>{{cite web| url=http://www.edvotek.com/300.051205.pdf |title=Blue/White Cloning of a DNA Fragment and Assay of β-Galactosidase |access-date=2023-09-10}}</ref>

====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.

{| class="wikitable"

|-

! 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&region=US |title= 5-Bromo-3-indolyl β-D-galactopyranoside |access-date=4 February 2014}}</ref>

|-

| 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 |access-date=4 February 2014}}</ref>

|-

| 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 |access-date=4 February 2014}}</ref>

|-

| 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&region=US |title=5-Bromo-6-chloro-3-indolyl-β-D-glucopyranoside |access-date=4 February 2014}}</ref>

|-

| Green-β-{{small|D}}-Gal || N-Methylindolyl-β-{{small|D}}-galactopyranoside || Green<ref>{{cite web|url=http://biotium.com/product/green-b-d-gal-n-methylindolyl-b-d-galactopyranoside/ |title=Green-β-D-Gal - Biotium, Inc. |access-date=4 February 2014}}</ref>

|-

| MUG, MUGA || 4-Methylumbelliferyl β-{{small|D}}-galactopyranoside || Fluorescent<ref>{{cite web|url=http://www.sigmaaldrich.com/catalog/product/sigma/m1633?lang=en&region=US |title=4-Methylumbelliferyl β-D-galactopyranoside |access-date=4 February 2014}}</ref> (λ_ex = 365, λ_em = 455)

|}

===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.<ref name="joung">{{cite journal

|vauthors=Joung J, Ramm E, Pabo C

|bibcode=2000PNAS...97.7382J

|doi-access=free

}}</ref> This technique may be limited to screening libraries of size of less than around 10⁶.<ref name="joung"/> The successful cleavage of X-gal also creates a noticeably foul odor due to the volatilization of [[indole]].

==See also==

* [[X-Gluc]]

[[Category:Chloroarenes]]

[[Category:Bromoarenes]]