5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside

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Structural formula
Structural formula X-Gal
General
Surname 5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside
other names
  • BCIG
  • X-Gal
Molecular formula C 14 H 15 BrClNO 6
Brief description

colorless crystalline solid

External identifiers / databases
CAS number 7240-90-6
EC number 230-640-8
ECHA InfoCard 100,027,855
PubChem 65181
Wikidata Q238475
properties
Molar mass 408.6 g mol −1
Physical state

firmly

Melting point

236 ° C (decomposition)

solubility

soluble in DMSO

safety instructions
GHS labeling of hazardous substances
07 - Warning

Caution

H and P phrases H: 302-312-332
P: 301 + 330 + 331-302 + 352
As far as possible and customary, SI units are used. Unless otherwise noted, the data given apply to standard conditions .

5-Bromo-4-chloro-3-indoxyl-β-D-galactopyranoside , also X-Gal , is an artificial glycoside and a chromogenic substrate for the enzyme β-galactosidase .

properties

The enzyme β-galactosidase hydrolyzes X-Gal to galactose and 5-bromo-4- chloroindoxyl (5-bromo-4-chloro-3-hydroxyindole). The 5-bromo-4-chloro-indoxyl is oxidized by the oxygen in the air to the deep blue dye 5,5'-dibromo-4,4'-dichloro- indigo .

Enzymatic cleavage of the X-Gal leads to the formation of 5-bromo-4-chloro-indoxyl ( 1 ). After oxidation in air and dimerization, this forms the blue dye 5,5'-dibromo-4,4'-dichloro-indigo ( 2 ).

use

In biochemistry , X-Gal is used for the qualitative determination of the activity of β-galactosidase. In contrast, the substrate o-nitrophenyl-β-D-galactopyranoside (ONPG) is used for quantitative determination .

Blue-white screening after transformation (LB agar with ampicillin and X-Gal)

In molecular biology , X-Gal is used for blue-white selection . In many cloning vectors is in the amino terminal coding sequence of β-galactosidase (α-fragment) a multiple cloning site into the foreign DNA, such as. B. PCR fragments can be introduced. This disrupts the β-galactosidase gene sequence and the enzyme is no longer expressed. From this it follows that the X-Gal is no longer split and therefore no blue dye is formed. The colonies formed after the transformation then do not appear blue like those with an empty vector , but white.

X-Gal is often used in conjunction with IPTG (for the induction of the lac promoter , see also lac operon ) for the screening of blue colonies. It is also used for the screening of β-galactosidase reporter gene activities in transfections of eukaryotic cells and for the detection of β-galactosidase in immunology and histochemical applications.

Other chromogenic substrates:

  • 5-bromo-3-indolyl-β-D-galactopyranoside (Blue-Gal, Bluo-Gal)
  • 6-chloro-3-indolyl-β-D-galactopyranoside (Y-Gal, Red-Gal, Rose-Gal, Salmon-Gal)
  • 5-iodo-3-indolyl-β-D-galactopyranoside (Purple-Gal)
  • 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside (Magenta-Gal)
  • N -Methylindolyl-β-D-galactopyranoside (Green-Gal)

Other fluorogenic substrates:

  • 4-methyl-umbelliferyl-β-D-galactopyranoside (MUG) (λ ex  = 365 nm, λ em  = 455 nm)

See also

Individual evidence

  1. ↑ a b c d e data sheet 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, 98 +% from AlfaAesar, accessed on December 25, 2019 ( PDF )(JavaScript required) .

literature

  • Sambrook, J .; Maniatis, T .; Russel, DW: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press; 3rd edition (2001) ISBN 0-87969-577-3 .
  • Horwitz, JP et al. (1964). Substrates for Cytochemical Demonstration of Enzyme Activity. I. Some Substituted 3-Indolyl-β-D-glycopyranosides. J Med Chem. 7 (4), pp. 574-575; doi: 10.1021 / jm00334a044 .
  • Edwards, MJ, and Taylor, MF (1993). Substitution of DMSO for DMF as a Solvent for X-Gal. BioTechniques, 14, p. 234.