Question

What enzymes are used for probe synthesis? This question is for a Agriculture biotechnology class. Thank you!

Answer 1

Enzymes have been used as detection probes for years and are still commonly used, even with the upsurge of fluorescent probes, because of their simplicity, sensitivity and broad-spectrum applications. Enzyme probes, including horseradish peroxidase (HRP) and alkaline phosphatase (AP), are used to detect target proteins through chromogenic, chemiluminescent or fluorescent outputs.

Enzymatic probes have in biological research methods, including immunohistochemistry (IHC), immunoblotting and enzyme-linked immunosorbant assays (ELISAs).

Enzymes function as assay reporters by reacting with substrates to produce colored, fluorescent or chemiluminescent signals that can be measured. Substrates can be either soluble or precipitating.

Enzyme label	Substrates
Horseradish peroxidase (HRP)	3,3'-diaminobenzidine (DAB) 3,3',5,5'-tetramethylbenzidine (TMB) 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) o-phenylenediamine dihydrochloride (OPD)
Alkaline phosphatase (AP)	Combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) p-Nitrophenyl Phosphate (PNPP)
Glucose oxidase	Nitro blue tetrazolium chloride (NBT)
β-galactosidase	5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (BCIG or X-Gal)