Ortho-Nitrophenyl-β-galactoside

• 2-nitrophenyl - β -D-galactopyranoside
• ONPG
• Niphegal

189-192 ° C

Attention

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O -nitrophenyl- β -D-galactopyranoside (ONPG ) is an artificial glycoside and chromogenic substrate for β -galactosidases, such as the lacZ gene of Escherichia coli encoded enzyme.

Properties

Certain β -galactosidases hydrolyze ONPG to galactose and the yellow dye o -nitrophenol.

Use

In biochemistry and microbiology diagnostics ONPG is used for the qualitative and quantitative determination of the activity of β -galactosidases, see ONPG assay.

An excellent substrate of β -galactosidase is the disaccharide lactose. Similar to X-gal, which is used for the qualitative β - Galactosidasebestimmung, ONPG has a lactose analog, which is a chromogenic substrate for β -galactosidase.

In a reaction mixture resulting from the hydrolytic cleavage of the colorless ONPG galactose and the yellow colored o -nitrophenol. To obtain the reaction is pseudo first order, ONPG must be present in excess in the mixture. The intensity of the yellow color is then only dependent on the concentration of β - galactosidase, and the reaction time. By adding sodium carbonate ( shift of the pH - value in the alkaline range (pH 11) ), the reaction is stopped, as β -galactosidases, like most enzymes, high pH values ​​are inactive.

The amount of the resulting o- nitrophenol is determined by absorbance in a photometer at 420 nm. Of reaction time, the absorption value and a value for the used biomass (protein concentration, turbidity of the bacterial culture, etc.), a specific activity of β -galactosidase can be calculated.