Assay Method Information

Assay Name:  beta-Galactosidase Assay
Description:  Enzymatic activity of beta-galactosidase from Escherichia coli (1.2 x 10^-10 M) was measured using the colorimetric substrate ortho-nitrophenyl-b-galactoside ([ONPG]: 2500, 2000, 1500, 1000, 500, 250, 125 uM) in Z-Buffer (100 mM sodium phosphate at pH = 7, 10 mM KCl, 1 mM MgSO4) to which was added fresh 50 mM beta-mercaptoethanol at 30 °C. In a 96-well plate were combined a 5x stock solution of Z-buffer (containing 250 mM beta-mercaptoethanol) and 5x stock solutions of ONPG. Samples were adjusted to a final volume of 180 uL with water. Samples were equilibrated at 30 °C for 5 minutes on a Synergy H1 microplate reader. A 10x stock solution of enzyme (20 uL) was then injected into each well and the production of 2-nitrophenol was quantified at 410 nm over a period of 10 minutes.
Affinity data for this assay
 

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