This chapter discusses the conventional harvest and assay of transfected luciferase reporter activity, the use of co transfected reporter gene alkaline phosphatase to monitor changes in plate-to-plate transfection efficiency, and finally, a modified “minilysate” protocol that allows for simultaneous measurement of both luciferase reporter activity and assay of nuclear DNA-binding activity within the same plate of transfected cells. The firefly luciferase reporter gene is a highly versatile tool for the studies of transcriptional regulation. The reporter assay is extremely sensitive, reproducible, and compatible with internal control alkaline phosphatase reporters. These features allow for the measurements of low levels of transcription and correction for variations in transfection efficiency. Further, the use of nonradioactive reagents for detection is a significant advantage in an era where radioactive waste disposal is an issue. The rapid mRNA and protein turnover of luciferase in mammalian cells are properties that can be utilized to measure inducible enhancer activity by preformed DNA-binding proteins.
ASJC Scopus subject areas
- Molecular Biology