2-Nitrophenyl β-D-galactopyranoside (ONPG) is a widely used chromogenic substrate for β-galactosidase. It consists of a β-D-galactopyranoside moiety linked to a 2-nitrophenyl group, enabling convenient colorimetric detection of enzymatic activity.
Upon enzymatic hydrolysis by β-galactosidase, ONPG is cleaved to release o-nitrophenol (ONP), which produces a yellow-colored product under alkaline conditions. The intensity of the yellow color can be quantitatively measured by spectrophotometry, typically at 420 nm.
Due to its simplicity, sensitivity, and reliability, ONPG is extensively used in β-galactosidase activity assays, gene expression studies (lacZ reporter system), enzyme kinetics, and microbiological research.
2-Nitrophenyl β-D-galactopyranoside functions as a chromogenic substrate for β-galactosidase.During the enzymatic reaction:
The increase in absorbance is directly proportional to β-galactosidase activity.
ONPG is widely used for quantitative determination of β-galactosidase activity in biochemical and microbiological systems.Typical applications include:
The compound is commonly used in lacZ reporter gene systems to study gene expression.Applications include:
ONPG is frequently used in enzyme kinetics experiments involving β-galactosidase.Typical uses include:
The substrate is widely used in microbial identification and molecular biology experiments.Applications include:
| Parameter | Recommended Conditions |
| Detection Method | Colorimetric spectrophotometric detection |
| Detection Wavelength | 420 nm |
| Buffer Systems | Phosphate buffer or Tris-HCl buffer |
| Temperature | 25–37 °C |
| Assay Format | Spectrophotometric assays, microplate reader assays |
Optimization may be required depending on enzyme source and assay conditions.
Keep container tightly closed in a dry and well-ventilated place
Keep in a cool place
Recommended storage temperature 2 - 8 °C
Researchers working in the following fields may benefit from this compound:
What is ONPG used for?
It is commonly used as a chromogenic substrate for β-galactosidase activity assays and lacZ reporter systems.
What wavelength is used to detect ONPG hydrolysis?
The released o-nitrophenol is typically measured at 420 nm.
Why is ONPG widely used in molecular biology?
Because it provides a simple and quantitative method for detecting β-galactosidase activity and gene expression.
