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Product information "X-Gal / 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside"
X-Gal (5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid) is a chromogenic substrate for β-galactosidase. It produces a blue color that can easily be detected visually. X-Gal is used in the identification of lacZ + bacteria, especially for the detection of ß-galactosidase, which is expressed by the recombinant vectors. X-Gal is often used in conjunction with IPTG > (for the induction of the lac promoter (lac operon)), for the screening of blue colonies. It is also used for the screening of ß-galactosidase reporter gene activity in transfections of eukaryotic cells.
X-Gal is a substrate of ß-galactosidase, which releases by cleavage of the substrate 5-bromo-4-chloro-3-indoxyl (X). The enzymatically cleaved indoxyl is oxidized to the insoluble indigo. As a stock solution, for example, a 20mg/mL X-Gal solution in dimethylformamide can be prepared. For the use in LB / Amp - plates this stock solution is to be diluted 1 : 1000 (final concentration 20μg/mL) or 1: 500 (final concentration 40μg/mL). The heated medium should thereby be cooled to about 50°C. The amount of X-Gal may be reduced if an X-Gal solution in DMF is sprayed to the agar plates (already with bacterial colonies!). X-Gal is the substrate of choice for blue-white selection of recombinant bacterial colonies with the lac+ genotype.
Specifications:
Assay: min. 98% (HPLC)
Appearance: white to off-white powder
MP: 223°C
a20°C/D (1%, 50% DMF): -62° +/- 2°
Water (K.F.): max: 1%
C14H15BrClNO6
MW = 408.6 g/mol
Application:
- Blue/white colony screening to distinguish recombinant (white) from non-recombinant (blue) colonies. - Visualization of beta-galactosidase reporter gene expression in transfected eukaryotic cells. - Detection of beta-galactosidase activity in immunological and histochemical procedures.Source:
syntheticClassification:
Documents:
Safety Data SheetCertificate
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Accessory Items
IPTG is commonly used in cloning procedures that require induction of β-galactosidase activity. It is used together with X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) or Bluo-Gal (5-Bromo-3-indolyl-beta-D-galactopyranoside) in blue-white selection of recombinant bacterial colonies that induce expression of the lac operon in Escherichia coli. Mode of operation:While IPTG binds to the lacI repressor it is altering the conformation of the lacl repressor, which prevents the repression of the β-galactosidase coding gene lacZ. Thus IPTG is an inducer of the lac operon in bacteria for detection of lac+ colonies. Dissolve in water to e.g. 200mg/mL, sterilise by filtration and store in aliquots at -20°C. For detection of transformants, use in final concentration of 0.1mM up to 1mM in LB-Medium.
IPTG is commonly used in cloning procedures that require induction of β-galactosidase activity. It is used together with X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) or Bluo-Gal (5-Bromo-3-indolyl-beta-D-galactopyranoside) in blue-white selection of recombinant bacterial colonies that induce expression of the lac operon in Escherichia coli. Mode of operation:While IPTG binds to the lacI repressor it is altering the conformation of the lacl repressor, which prevents the repression of the β-galactosidase coding gene lacZ. Thus IPTG is an inducer of the lac operon in bacteria for detection of lac+ colonies. Dissolve in water to e.g. 200mg/mL, sterilise by filtration and store in aliquots at -20°C. For detection of transformants, use in final concentration of 0.1mM up to 1mM in LB-Medium.
Accessory Items
5-Bromo-4-chloro-3-indolyl-beta-D-glucopyranoside (X-glcA, CHX, X-Glu, X-Glc) was found to be the most suitable synthetic substrate for the demonstration of alpha-D-glucosidases activity in situ. X-Glu corresponds to X-Gal which is used as a substrate for beta-Galactosidase.Glucosidase releases 5-bromo-4-chloro-3-indoxyl (X) by cleavage of the substrate. The enzymatically cleaved indoxyl is oxidized by oxigen to insoluble indigo which forms a blue precipitate.Synonyms: 5-Bromo-4-chloro-3-indolyl β-D-glucopyranoside; 5-Bromo-4-chloro-3-indoxyl beta-D-glucopyranoside; X-glucoside; X-b-D-Glucoside; X-β-D-Glucoside.
Rose-Gal, also known as Red-Gal or Salmon-Gal, (6-Chloro-3-indolyl-ß-D-galactopyranoside), is a is a beta-galactosidase substrate which produces a salmon pink colour. Rose-Gal (Red-Gal / Salmon-Gal) serves as an alternative to X-Gal > for the enzymatic detection of Lac+ beta-Galactosidase activity in bacterial colonies. In colorimetric assays, recombinants (white) can be distinguished from non-recombinants (salmon). In conjunction with X-Gluc >, Rose-Gal (Red-Gal, Salmon-Gal) can be used for the simultaneous detection of GUS and Lac + activity on the same plate.
X-alpha-D-Gal is a chromogenic substrate for alpha-galactosidase yielding a blue precipitate (X-alpha-Gal is a chromogenic substrate for yeast galactosidase (MEL1) and is used for detecting GAL4-based yeast two-hybrid interactions directly on agar). Used for species differentiation within the family Enterobacteriaceae and differentiation of Bifidobacteria species from Lactobacillus species (see references). Synonmys: 5-Bromo-4-chloro-3-indolyl a-D-galactopyranoside, X-α-Gal; X-alpha-Gal. Other products that are used as a substrate for alpha or beta-Galactosidase: M3225: 5-Bromo-3-indolyl-beta-D-galactopyranoside >M3204: 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) >M3205: 5-Bromo-4-chloro-3-indolyl-ß-D-glucopyranoside (X-Glu / X-Glc) > References: 1. Aho S., Arffman A., Pummi T., Uitto J., A novel reporter gene MEL1 for the yeast two-hybrid system. Anal Biochem. 1997, 253, 270-2. 2. Chevalier P., et al., X-alpha-Gal-base medium for simultaneous enumeration of bifidobacteria and lactic acid bacteria in milk. J. Microbiol. Methods 1991, 13, 75. 3. Gossrau R., Lojda Z., Histochemical detection of alpha-D-galactosidase with 5-Br-4-Cl-3-indoxyl alpha-D-galactoside. Acta Histochem. 1989, 85, 213-22. 4. Perry J.D., Ford M., Taylor J., Jones A.L., Freeman R., Gould F.K., ABC medium, a new chromogenic agar for selective isolation of Salmonella spp. J. Clin. Microbiol. 1999, 37, 766-8.