Firefly&Renilla-Glo Luciferase Reporter Assay Kit，IC-6060

Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology. Applications include the study of receptor activity, transcription factors, intracellular signaling, mRNA processing and protein folding. Dual reporters are commonly used to improve experimental accuracy. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system. Typically, the “experimental” reporter is correlated with the effect of specific experimental conditions, while the activity of the co-transfected “control” reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by differences in cell viability or transfection efficiency. Other sources of variability, such as differences in pipetting volumes, cell lysis efficiency and assay efficiency, can be effectively eliminated. Thus, dual-reporter assays often allow more reliable interpretation of the experimental data by reducing extraneous influences.

The Firefly & Renilla Luciferase Reporter Assay System(a-c) provides an efficient means of performing dual-reporter assays. In the Firefly & Renilla Luciferase Reporter Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement. In the Firefly & Renilla Luciferase Reporter Assay System, both reporters yield linear assays with subattomole sensitivities and no endogenous activity of either

  A. The Firefly & Renilla Luciferase Reporter Assay

 Firefly and Renilla luciferases, because of their distinct evolutionary origins, have dissimilar enzyme structures and substrate requirements. These differences make it possible to selectively discriminate between their respective bioluminescent reactions. Thus, using the DLR™ Assay System, the luminescence from the firefly luciferase reaction may be quenched while simultaneously activating the luminescent reaction of Renilla luciferase. Firefly luciferase is a 61kDa monomeric protein that does not require post-translational processing for enzymatic activity (1,2). Thus, it functions as a genetic reporter immediately upon translation. Photon emission is achieved through oxidation of beetle luciferin in a reaction that requires ATP, Mg2+ and O2 (Figure 1). Under conventional reaction conditions, the oxidation occurs through a luciferyl-AMP intermediate that turns over very slowly. As a result, this assay chemistry generates a “flash” of light that rapidly decays after the substrate and enzyme are mixed. Many of our Luciferase Assay Reagents for quantitating firefly luciferase incorporate coenzyme A (CoA) to provide more favorable overall reaction kinetics (3). In the presence of CoA, the luciferase assay yields stabilized luminescence signals with significantly greater intensities (Figure 2) than those obtained from the conventional assay chemistry. The firefly luciferase assay is extremely sensitive and extends over a linear range covering at least seven orders of magnitude in enzyme concentration (Figure 3). Renilla luciferase, a 36kDa monomeric protein, is composed of 3% carbohydrate when purified from its natural source, Renilla reniformis (4). However, like firefly luciferase, post-translational modification is not required for its activity, and the enzyme may function as a genetic reporter immediately following translation. The luminescent reaction catalyzed by Renilla luciferase utilizes O₂ and coelenterate-luciferin (coelenterazine; Figure 1).

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