Luciferin is the basic substrate of bioluminescent assays. Based on a simple reaction where ATP fuels luciferase catalysis of firefly (beetle) luciferin to produce light, bioluminescence has broad applications. Its employment as a biomass indicator allows for the detection of microorganisms in consumer products, manufacturing plant surfaces, and has even been used to determine if life exists on the moon and Mars.
Bioluminescence is being used in in vitro and in vivo monitoring of biological processes including gene expression and protein-protein interactions. Luciferin is used, for example, in reporter gene assays to study gene regulation and function where expression of the luciferin-tagged reporter is a marker to indicate successful uptake of the gene of interest in recombinant DNA techniques. As a detection reagent, the luciferin-luciferase reaction is utilized in pyrosequencing to accomplish fast detection of bases in today’s high-throughput DNA sequencing systems.
More recently, bioluminescence imaging has emerged as a powerful technique for the direct study of different cell populations in live small animals such as mice. In this technique, cells (e.g., cancer cells, stem cells, T cells) are designed to express luciferase and illuminate. Noninvasive visualization in live animals using a sensitive charge-couple device camera thus makes it possible for real-time observation of disease progression and regression at different times over the course of therapeutic treatment. Employed to understand the molecular basis of pathologies such as neurodegenerative disorders, cardiovascular disorders, obesity, and cancer, this technique is of immense value for clinical, diagnostic, and drug development.
Scientists at Regis Technologies have identified common weaknesses in luciferase assays. Problems often occur before the assay begins. Firefly luciferin is a sensitive molecule, and even well-planned experiments can give erroneous results if the luciferin is mishandled and damaged before use. In this article, we describe the optimal salt, purity, handling, and storage of luciferin to ensure successful and consistent results in animal imaging and other assays.