KINETIC DETERMINATION OF CELLULAR LACZ EXPRESSION

A kinetic assay for the expression of beta-galactosidase in cells tran sfected with the LacZ gene was developed using a 96-well-plate for mat . 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 temper atures, thus permitting a semiautomated or fully automated determinati on of reporter expression. The rates of chromophore development were f ound to be linear over a 6-log enzyme concentration range, from 0.001 to 100 mU. Additionally, the use of kinetic data avoids the complicati ons of non-enzymatic, background optical density.