Dual Luminescence-Based Reporter Gene Assay for Luciferase andβ-Galactosidase

Abstract

A unique combined luminescence assay for firefly (Photinus pyralis) luciferase and β-galactosidase (β-gal) reporter gene products is described. Luciferase and β-gal activities are determined with the same aliquot of cell lysate prepared from cells contransfected with both reporter genes, thereby reducing manual labor and increasing experimental accuracy. With the Dual-LightTM assay system, luciferase activity is measured first with an enhanced luciferase assay, followed by quantitation of β-gal with Galacton-PlusTM chemiluminescent substrate and Sapphire-IITM enhancer. Highly sensitive detection of luciferase (2 fg) and β-gal (8 fg) is achieved with a dynamic range over seven orders of magnitude of enzyme concentration. Comparative analysis of both independent and combined (Dual-Light) detection methods for cells contransfected with luciferase and β-gal reporter genes is also described.