Screening Reagents

Tetrazolium red is used to visualize dehydrogenase enzyme activity. Initially, the tetrazolium solution is colorless but changes to red when it comes into contact with hydrogen. Tetrazolium red is used in a biochemical viability test for seeds. The test relies on dehydrogenase enzymes to release hydrogen ions, which subsequently reduce the colorless tetrazolium salt solution to a red compound called formazan. Living cells turn red while dead cells remain colorless.

4-Methylumbelliferyl caprylate is a fluorogenic substrate for esterase with C8 activity, yielding a blue fluorescent solution upon cleavage and is used for the identification of Salmonella spp. based on the detection of caprylate esterase.

4-Nitrophenyl-α-D- galactopyranoside is a chromogenic substrate yielding a soluble chromophor for the colorimetric assay of alpha-D-galactosidase upon cleavage. Used for the detection of hydrolases in tissues and membranes.

4-Nitrophenyl-α-D- glucopyranoside is a chromogenic substrate for alpha-D-glucosidase, yielding a yellow solution upon cleavage. 4-Nitrophenyl-α-D- glucopyranoside is also used for the detection of glucansucrases and for yeast alpha-D-glucosidase and for crystal structure studies of the complexes of concanavalin A.

X-butyrate can be used as a substrate for carboxyl esterase, yielding a blue precipitate upon cleavage.

5-Bromo-4-chloro-3-indoxyl octanoate; X-caprylate

MagentaTM-butyrate

Bluo-Gal is used as an alternative to X-Gal, and produces a darker blue color. Bluo-Gal is hydrolyzed by β-galactosidase to produce a blue precipitate. This precipitate is insoluble in alcohol and xylenes, thus making it suitable for immunoblotting and immunocytochemical assays. Bacterial colonies containing active β-galactosidase produce blue colonies when grown on plates containing Bluo-Gal.

Yellow crystalline powder that is used for the colorimetric determination of thiol groups in biological samples. DTNB is fairly soluble in water.

IPTG is an analog of galactose that is non-metabolizable and inactivates the lac repressor to induce synthesis of β-galactosidase in E. coli. The expression of cloned genes under the control of the lac operon is induced by IPTG. It is also a substrate for thigalactoside transacetylase and has been reported to induce penicillinase in bacteria.

The IPTG EZ Pak™ is the fastest and easiest way to make a set amount of sterile IPTG solution. The kit includes preweighed IPTG powder, a sterile filter, and a sterile container for the filtered solution. No need to calculate, simply add the stated amount of deionized H₂O, filter, and pour into the labeled bottle for easy usage. The EZ Pak™ includes high quality GoldBio IPTG and the sterile solution is ready for tissue culture, bacterial media, or any other appropriate use.

Magenta-Gal is used as an alternative to X-Gal, and produces a red color. Magenta-Gal is hydrolyzed by β-galactosidase to produce a red precipitate. This precipitate is insoluble in alcohol and xylenes, thus making it suitable for immunoblotting and immunocytochemical assays. Bacterial colonies containing active β-galactosidase produce red colonies when grown on plates containing Magenta-Gal.

Magenta-Gal is commonly used in conjunction with IPTG (GoldBIo #I2481) for red/white screening.

4-Methylumbelliferyl beta-D-glucuronide (MUG) is a fluorogenic substrate for beta-glucuronidase (GUS). Upon cleavage, MUG yields blue fluorescence seen under UV light.

This substrate is commonly used for the detection of Escherichia coli through its ability to detect activity of beta-glucuronidase, which is encoded by uidA. GUS activity is tested in minimal medium o-nitrophenyl-beta-D-galactopyranoside (MMO)-MUG.

It is ideal for plant molecular study as a way to examine gene expression under various promoters. And while it works well for measuring activity in plant lysate and leaf disks, it can also work in whole plant assays.

Chromogenic substrate for beta-D-galactosidase, yielding a green precipitate upon cleavage

Nitrotetrazolium Blue (NBT) is used in combination with 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) to detect alkaline phosphatase activity in blotting techniques and in situ hybridization chemistry. BCIP acts as the substrate for alkaline phosphatase, and the oxidizing reagent NBT enhances the purple-brown final color of the precipitates at the reaction sites.

NBT has also been used with X-Gal,or Salmon-Gal to speed up and enhance colony screening.

Salmon Gal (6-Chloro-3-indolyl-β-D-galactopyranoside) is used with IPTG as an alternative chromogenic substrate capable of detecting LacZ gene encoded ß-galactosidase activity for colony screening in colometric assays.

Unlike colony blue/white screening through X-Gal, nonrecombination results in a salmon color after salmon gal is cleaved by ß-galactosidase through hydrolysis. This contrast may be useful in comparative or secondary screenings.

Incorporating nitroblue tetrazolium salt (NBT) into the salmon gal reactions provides faster results with a clear, purple precipitate than in unaccompanied uses of salmon gal.

TMB is an excellent colorimetric substrate for detection of horseradish peroxidase labeled probes and is used with peroxidase and peroxidase-coupled systems, particularly in ELISA techniques. It produces a soluble end product, which is blue in color and can be read spectrophotometrically. TMB has also been used in the quantitative determination of hemoglobin and in cytochemical staining for peroxidase. TMB is a hepatitis B antigen.