ONE-Glo ™ Luciferase Assay System: New Substrate, Better Reagent (2007)

Early luciferase assay reagents required removal of assay components such as medium, serum and test compounds before measuring luminescence.The advent of homogeneous luciferase assays simplified reactions by eliminating the need for media removal prior to luminescence measurement and by enabling light measurement over an extended time period. Now a novel luciferin analog has been developed that enables luciferase assay reagents to operate at a lowered pH.This simple change leads to a reagent that is easier to use, more robust to the reaction environment and less aromatic than reagents containing unmodified luciferin. By Er ika M. Hawkins1, Michael P. Valley1, Mike A. Scurr ia2, James Unch2, Troy Good2, Dave Good2, Laurent Bernad2, Dieter H. Klauber t2, Keith V. Wood1, 1Promega Corporation and 2Promega Biosciences ONE-GloTM Luciferase Assay System: New Substrate, Better Reagent LUCIFERASE ASSAY NUMBER 97 SEPTEMBER 2007 WWW.PROMEGA.COM 30 INTRODUCTION Firefly luciferase assays have been used for more than 15 years to quantitate reporter gene expression. During this period, improvements in both luciferase genes and luciferase assay reagents have made luciferase assays one of the simplest reporter technologies available, both to implement and to read. Now a novel luciferin substrate analog has been developed. Based on this substrate, the ONE-GloTM Luciferase Assay System(a,b,c) is a unique, homogeneous luciferase reagent that is more stable, more robust and less aromatic than standard luciferin reagents. Luciferase enzyme expression is often quantified in studies ranging from signal transduction to protein:protein interactions, transcription and a wide assortment of other assay types (1,2). A monomer that requires no post-translational modification, firefly luciferase is well suited to these tasks. Furthermore, with the pGL4 Luciferase Reporter Vectors Promega improved the luciferase gene as a biological tool by optimizing mammalian expression, minimizing off-target responses and actively controlling protein half-life (3,4). Quantifying luciferase in high-throughput and ultrahigh-throughput environments requires a highly sensitive and robust reagent. Major improvements in luciferase reagent technology were made when homogeneous reagents were introduced. These early homogeneous reagents contained inhibitors to slow the luciferase reaction and thus prolong the luminescent signal. All subsequent reagents have been variations on this theme (5–7). These reagents work well but suffer limitations inherent in the luciferin chemical reaction and in the additives necessary to stabilize the luminescent signal. We have created a new, proprietary reagent that contains a luciferin substrate analog, 5 -fluoroluciferin(a) (Figure 1). The addition of a fluorine at the 5 position improves some of the fundamental characteristics of the luciferase reagent. Luciferase assays made with fluoroluciferin have lower thiol concentrations than conventional luciferase reagents and thus less thiol-associated odor. In addition, the fluoroluciferin-containing components and reconstituted reagents are much more functionally stable than similar luciferin-based reagents. Due to the presence of fluoroluciferin, the ONE-GloTM Reagent is also more compatible with other compounds within the luciferase reaction, such as phenol red and small molecules that may inhibit luciferase. IMPROVED COMPONENT AND REAGENT STABILITY One of the unique characteristics of luciferase reactions containing fluoroluciferin is that the pH optimum for the reaction is much lower than for reactions containing conventional luciferin. Although the pH optimum of the luciferase-luciferin reaction is approximately 7.8 (8), pH 7.0 is optimal for the luciferase-fluoroluciferin reaction (Figure 2). In order to be useful as a substrate for luciferase, the 6 -OH of luciferin must be deprotonated. The fluorine in 5 -fluoroluciferin acts as an electron acceptor and lowers the pK of the 6 -OH to allow it to remain predominantly deprotonated at neutral pH. A novel luciferin substrate analog has been developed to improve luciferase reagent technology.