pGL3 Luciferase Reporter Vectors Technical Manual #TM033

VI.Assay of Luciferase Activity (continued)The Luciferase Assay System has long been the standard reagent for routinelaboratory analysis. Before using this reagent, cells from which the luciferase isto be measured must be washed and lysed. This reagent was optimized forhigh sensitivity in nonhomogeneous, single-sample measurements. TheLuciferase Assay System requires a luminometer fitted with injectors toefficiently measure luminescence in 96-well plates.The Bright-Glo, Steady-Glo ® and ONE-Glo Reagents were developed toperform assay reactions within multiwell plates and in the presence ofcomplete cell culture medium: no cell preparation steps such as washing orlysing are required before the luminescence reaction is initiated. All of these aresingle-step reagents, requiring only addition of the reagent before measuringluminescence. This makes them ideal reagents for efficient and precisequantitation in 96-, 384- and 1536-well plates.The Bright-Glo and Steady-Glo ® Reagents are complementary in theircharacteristics based on the inverse relationship between luminescenceduration and assay sensitivity (6). Generally, as the half-life of the luminescenceincreases, assay sensitivity decreases. The Steady-Glo ® Reagent provides longluminescence duration (changing only about 10% per hour); however, toachieve this long luminescence duration, the assay sensitivity must be reduced.This reagent was designed for experiments in which many microplates areprocessed as a batch.In contrast, the Bright-Glo Reagent provides high assay sensitivity withshorter luminescence duration (