Kinetic determination of cellular LacZ expression

A kinetic assay for the expression of beta-galactosidase in cells transfected with the LacZ gene was developed using a 96-well-plate format. The assay involves solubilization of the cells followed by measuring hydrolytic rates of o-nitrophenyl beta-D-galactoside on a standard 96-well-plate reader without other manipulations. The protocol requires only that reagent be added sequentially to the wells at ambient temperatures, thus permitting a semiautomated or fully automated determination of reporter expression. The rates of chromophore development were found to be linear over a 6-log enzyme concentration range, from 0.001 to 100 mU. Additionally, the use of kinetic data avoids the complications of non-enzymatic, background optical density.