Choosing a detection assay depends on several factors. As shown earlier, your choice of reporter will affect signal strength, response time and sensitivity. If you choose Nluc or NlucP as your reporter, you will detect activity with the Nano-Glo® Luciferase Assay System, which provides stable, glow-type signal in the presence of Nluc, with a half-life of approximately 120 minutes.
In many cases, firefly luciferase is still a very good reporter option and many constructs exist using Fluc and FlucP as the reporter. There are multiple assay chemistry options for detection of the Fluc reporter allowing you to choose the detection assay most appropriate for your particular experimental needs. Assays for detection of firefly luciferase include options with either “flash” or “glow” signal kinetics. A “flash” assay is one where the signal is strong but short-lived requiring injection-based delivery of reagent. In contrast, a “Glow” type assay has a prolonged signal eliminating the need for injectors, but with generally lower signal intensities. Learn more about “flash vs glow”. The figure below compares signal strength and duration using different firefly assays with glow signal kinetics. The Bright-Glo™ Assay provides the brightest signal, but it decays rapidly. On the other hand, the Steady-Glo® Assay provides the most consistent signal duration with the lowest intensity. The ONE-Glo™ and ONE-Glo™ EX assays are ideally suited for high-throughput use. The ONE-Glo™ EX Reagent has the added advantage of improved stability, allowing you to make up the reagent and store it at 4⁰C for several weeks, simplifying repeat use if you have a set of experiments to run.
Figure 4. Comparison of five single glow kinetic firefly luciferase reporter assays. One hundred microliters of purified firefly luciferase (13.8ng/ml in DMEM with 0.1% Prionex® as carrier) was combined in a 96-well plate with 100µl of Bright-Glo™, ONE-Glo™, ONE-Glo™ EX, Dual-Glo® Luciferase or Steady-Glo® Reagents. Luminescence was measured periodically over 2 hours, n = 8.