Changes

β-D-galactopyranoside, C19H21O9Na, MW 416.35,  MDL Number MFCD03458481.

β-D-galactopyranoside, C19H21O9Na, MW 416.35,  MDL Number MFCD03458481.

S-Gal®, sodium salt is a patented water-soluble, autoclavable chromogenic substrate for β-galactosidase that is designed to replace X-Gal in blue-white selection of recombinant bacterial colonies with the lac+ phenotype. Caution: For black color development to occur, ferric ion must be present. Although we recommend ferric ammonium citrate (500 mg/L of media), other ferric compounds can be used to provide this requirement, depending upon your particular system.  From [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=S7313%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC Sigma-Aldrich site].

S-Gal®, sodium salt is a patented water-soluble, autoclavable chromogenic substrate for β-galactosidase that is designed to replace X-Gal in blue-white selection of recombinant bacterial colonies with the lac+ phenotype. Caution: For black color development to occur, ferric ion must be present. Although we recommend ferric ammonium citrate (500 mg/L of media), other ferric compounds can be used to provide this requirement, depending upon your particular system.  From [https://www.sigmaaldrich.com/US/en/product/sigma/s7313 Sigma-Aldrich site].

[[Image:sgal.tiff|400px|left]]

[[Image:sgal.png|400px|left]]

Image from Heuermann and Cosgrove PMID 11355350

Image from Heuermann and Cosgrove PMID 11355350

==S-Gal staining conditions==

==S-Gal staining conditions==

According to [http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=S7313%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC Sigma-Aldrich site]:  

According to [https://www.sigmaaldrich.com/US/en/product/sigma/s7313 Sigma-Aldrich site]:  

Stock solutions of S-Gal can be made by dissolving at 50 mg/mL in deionized water, sterile-filtering and storing at -20 °C.

Stock solutions of S-Gal can be made by dissolving at 50 mg/mL in deionized water, sterile-filtering and storing at -20 °C.

S-Gal, IPTG and ferric ammonium citrate are dry-blended with standard LB agar at the final concentrations stated above. The powdered blend was suspended in deionized water and autoclaved as previously described. For the preparation of microwaved medium, the suspended blend was heated until boiling, followed by swirling to allow the remaining agar component to go into solution. In this case, the antibiotic ampicillin was added after autoclaving (100 mg/mL).  

S-Gal, IPTG and ferric ammonium citrate are dry-blended with standard LB agar at the final concentrations stated above. The powdered blend was suspended in deionized water and autoclaved as previously described. For the preparation of microwaved medium, the suspended blend was heated until boiling, followed by swirling to allow the remaining agar component to go into solution. In this case, the antibiotic ampicillin was added after autoclaving (100 mg/mL).

 

=X-Gal=

=X-Gal=

http://www.bio.davidson.edu/courses/genomics/method/xgal.gif

https://bio.davidson.edu/courses/genomics/method/xgal.gif

Cleavage by beta-galactosidase yields a dihalogen-substituted indoxyl, 5-bromo-4-chloro-3-indoxyl:

Cleavage by beta-galactosidase yields a dihalogen-substituted indoxyl, 5-bromo-4-chloro-3-indoxyl:

Oxidation in the presence of an electron acceptor yields a relatively insoluble indigoid dye:

Oxidation in the presence of an electron acceptor yields a relatively insoluble indigoid dye:

=Oxidants used in histochemistry=

=Oxidants used in histochemistry=

Performic acid PMID: 1099403

Performic acid PMID: 1099403

Cepko, C., Ryder, E., Fekete, D.M., and Bruhn, S. Detection of B-galactosidase and alkaline phosphatease activities in tissue. Online: http://genepath.med.harvard.edu/~cepko/protocol/xgalplap-stain.htm. An excellent brief discussion of X-Gal histochemistry. Much of the material presented here was derived using their references.  

Cepko, C., Ryder, E., Fekete, D.M., and Bruhn, S. Detection of B-galactosidase and alkaline phosphatease activities in tissue. Online (archive): https://web.archive.org/web/20160821210733/http://genepath.med.harvard.edu/~cepko/protocol/xgalplap-stain.htm. An excellent brief discussion of X-Gal histochemistry. Much of the material presented here was derived using their references.

=Studies in Enzyme Cytochemistry=

=Studies in Enzyme Cytochemistry=

Recommends ferrocyanide, and notes that it inhibits red blood cell staining "presumably because of the chemical similarity to cyanide"

Recommends ferrocyanide, and notes that it inhibits red blood cell staining "presumably because of the chemical similarity to cyanide"