2-Nitrophenyl β-D-galactopyranoside

Product No. N1127

CAS RN 369-07-3
EC 206-716-1
Synonyms: ONPG

Product Description

This product (ONPG) is a colorimetric substrate for β-galactosidase. This product is hydrolyzed to o-nitrophenol by the enzyme. o-Nitrophenol is yellow in alkaline solution and can be quantitated at 410-420 nm.1 The molar extinction coefficient is 2.13 x 104 at pH 10.2.3 We have used absorbance readings at 410 nm in the determination of the activity of β-galactosidase using this product as the substrate. The Km for β-galactosidase (from E. coli) is 9.5 x 10-4 M in Tris buffer, pH 7.6, at 20 °C.3 The Vmax for β-galactosidase for this substrate is 134 µmoles/min/mg protein.4

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

Preparation Instructions

A stock solution of this product can be prepared in deionized water at a concentration of 3 mg/ml. Alternatively, a stock solution may be prepared in phosphate buffer. When used as a substrate for β-galactosidase, a solution of approximately 20.5 mg/ml is prepared in 100 mM sodium phosphate buffer, pH 7.3. The solution is gently warmed to completely dissolve the product.

Product Profile

Molecular Formula: C12H15NO8
Molecular Weight: 301.3
Melting Point: 193-194 °C 5
Specific Rotation: -67° (1 g/100 ml water at 25 °C)




  1. Molecular Cloning: A Laboratory Manual, 2nd ed., 3, Sambrook, J., et al., Cold Spring Harbor Laboratory (1989), p. 1666.
  2. Adv. Carbohydr. Chem., 16, 239 (1961).
  3. Enzyme Handbook, II, Barman, T. E., Springer-Verlag (Berlin-Heidelberg: 1969), p. 581.
  4. Wallenfels, K., b-Galactosidase (Crystalline). Methods in Enzymology, 5, 212-218 (1962).
  5. J. Am. Chem. Soc., 75, 1758 (1953).


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