onitrophenylDgalactopyranoside

O-nitrophenyl-β-D-galactopyranoside (ONPG) is a chromogenic substrate widely used in biochemistry and molecular biology to measure beta-galactosidase activity. It is a colorless glycoside that, when cleaved by beta-galactosidase, yields galactose and o-nitrophenol, a compound that develops a yellow color under alkaline conditions. ONPG is commonly employed in liquid assays rather than plate assays, providing a quantitative readout of enzyme activity.

The enzymatic reaction involves hydrolysis of the glycosidic bond by beta-galactosidase, releasing o-nitrophenol. The intensity of the yellow color correlates with the amount of product formed and thus with enzyme activity. In practice, the assay is performed by incubating a sample containing beta-galactosidase with ONPG at an appropriate temperature, often around 37°C, and then stopping the reaction with a basic stop solution to maximize color development. Absorbance is typically measured at 420 nm to quantify o-nitrophenol formation. Resulting activity is frequently reported in units such as Miller units, which provide a standardized way to compare samples.

ONPG is a standard tool in lacZ reporter assays and related studies, where beta-galactosidase activity reflects promoter activity or gene expression. It offers a convenient, spectrophotometric alternative to chromogenic substrates like X-gal, which produce insoluble colored precipitates. Preparation and storage follow typical laboratory reagent guidelines, with attention to stability and safety. Consult the product’s data sheet for specific concentrations, incubation times, and disposal requirements.