ONPG (2-Nitrophenyl β-D-galactopyranoside)

A chromogenic substrate for β-galactosidase

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1g

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货号: ajce51104
CAS: 369-07-3
别名: 2-硝基苯-beta-D-半乳糖苷,2-Nitrophenyl β-D-galactopyranoside
分子式: C12H15NO8
分子量: 301.25
纯度: >98%
溶解度: Water : 7.4 mg/mL (24.56 mM)
储存: Store at -20°C
库存: 现货

Background

o-Nitrophenyl β-D-galactopyranoside (ONPG) is a chromogenic substrate for β-galactosidase.^1,2 Upon enzymatic cleavage by β-galactosidase, o-nitrophenol is released, which can be quantified by colorimetric detection at 420 nm as a measure of β-galactosidase activity.

1.Serebriiskii, I.G., and Golemis, E.A.Uses of lacZ to study gene function: Evaluation of β-galactosidase assays employed in the yeast two-hybrid systemAnal. Biochem.285(1)1-15(2000) 2.Li, W., Zhao, X., Zou, S., et al.Scanning assay of β-galactosidase activityPrikl. Biokhim. Mikrobiol.48(6)668-672(2012)

Protocol

Kinase experiment:

The β-galactosidase activity is measured using two substrates including ONPG and lactose in this study. The β-galactosidase activity for ONPG is measured by following the amount o-nitrophenol released from ONPG. The reaction mixture is composed of 100 μL of the enzyme solution and 400 μL of ONPG solution (2.5 g/L in 100 mM Tris-HCl buffer at pH 6.8). After incubation at 78°C for 15 min, the reaction is terminated by adding an equal volume of 1 M Na2CO3. The released o-nitrophenol is quantitatively determined by measuring at A405. One unit of activity is defined as the amount of enzyme needed to produce 1 μmol of o-nitrophenol per minute under the assay condition. The specific activity is expressed as units per milligram of protein. Assays for activity towards lactose are performed in the same buffer containing 100 μL of enzyme solution and 5% lactose, and the reaction is stopped by boiling for 10 min, and the concentration of glucose is determined using a glucose oxidase-peroxidase assay kit. The released glucose is quantitatively determined by measuring A492. One unit of enzyme activity is defined as the amount of activity required to release 1 μmol of glucose per minute[1].

参考文献:

[1]. Zhang X, et al. Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin. BMC Microbiol. 2013 Oct 24;13:237. doi: 10.1186/1471-2180-13-237.