The Thermo Scientific Pierce Renilla-Firefly Luciferase Dual Assay Kit provides the necessary reagents to simultaneously detect intracellular Renilla and Red Firefly luciferase activity in mammalian whole cell lysates.
The Pierce Renilla-Firefly Luciferase Dual Assay Kit is a highly sensitive assay that allows for the simultaneous detection of Green Renilla and Red Firefly luciferase activity. Green Renilla luciferase acts as an experimental reporter with constitutively active red firefly as a normalization control. As shown in the animation video below, this reporter-and-control combination enables simultaneous monitoring of experimental reporter and control luciferase activities in a single-read assay without the need for two-step addition of substrate reagents or quenching. The assay working solution contains substrates for both luciferases, and the reactions occur simultaneously with flash-type kinetics. The resulting luminescent signals are spectrally resolvable using filters. In a single sample, researchers can assay transcriptional activity of regulatory elements, signal transduction pathways, and effects of activators or inhibitors.
- Simultaneous – concurrent, filter-based, wavelength-separated detection of two luciferase activities
- Sensitive – measure Green Renilla and red firefly luciferase activities in the same sample
- Fast – no quenching step required, unlike in traditional sequential dual assays
- Multiplex – capable of quantitating two cellular activities in the same sample(s)
Cell lysis buffer, reaction buffer, and substrates coelenterazine and D-luciferin
Green Renilla and Red Firefly luciferase reporters; Filter sets at 525nm±20nm for Green Renilla Luc and 615 to 675nm for Red Firefly Luc. Luminometer or other instrument capable of monitoring luminescence, such as Thermo Scientific Luminoskan Ascent and Varioskan Flash Microplate Readers; Injector required for assessing more than 24 wells at a time.
- Study two regulatory elements at the same time
- Monitor two signaling pathways simultaneously
- Enable studying more than one target per screen (e.g. off-target effects)
Watch this video animation to learn about performing dual-spectral luciferase assays.
Green Renilla produces brighter bioluminescent signal than firefly and native Renilla luciferases. The bioluminescent signal (λmax= 535nm) produced by Green Renilla luciferase protein results from the oxidation of coelenterazine. The second assay uses red firefly luciferase, which is a mutant form of the Japanese firefly luciferase Luciola cruciata. This luciferase produces a red-shifted emission spectrum (λmax= 613nm) that results from the oxidation of D-luciferin in the presence of ATP.
|Thermo Scientific Pierce Renilla-Firefly Dual Luciferase Assay emission spectra profile.Shaded ranges represent the filter sets used in product development experiments: 525±20nm band pass (BP) and 640nm long pass (LP).
|Protocol summary for the Thermo Scientific Pierce Renilla-Firefly Dual Luciferase Assay Kit. This is a one-step procedure for spectrally resolving two luciferases. By including both substrates in a single assay reagent, two luciferase activities can be measured simultaneously in a sample by filter-based detection.
- Shimomura, O. (2006). Bioluminescence: Chemical Principles and Methods. World Scientific Publishing Co. Pte. Ltd, Hakensack NJ.
Review of Luciferase Reporters
Luciferase Reporter Assay Selection Guide
Dual-spectral Luciferase Luminescence Calculator
Firefly Vectors, Flash and Glow Assay Kits
Renilla Vectors, Glow and Dual Assay Kits
All Luciferase Reporter Assay Reagents and Kits
Pierce Luciferase Cell Lysis Buffer (2X)
TurboFect in vitro Transfection Reagent
Modified Dulbecco’s PBS BupH Packs
Modified Dulbecco’s PBS, 20X Solution