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.
Marsh
J
J
Unit on Lymphocyte Function, Laboratory of Molecular Biology, NIMH, Bethesda, Maryland 20892.
D001681:Biological Assay / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D007700:Kinetics; D007763:Lac Operon; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
D001616:beta-Galactosidase