Luciferase Assay Selection Guide
Thermo Scientific Pierce Luciferase Reporter Assay Kits allow you to investigate promoter activity by measuring light output from luciferase enzyme that is expressed under the control of your promoter of interest.
Luciferase genes vary in stability, light output and spectral characteristics, and reporter kits are available with different luciferases for both single- and dual-spectral analyses. This guide will help you determine which luciferase enzymes and kits are most appropriate for your experiment.
The Thermo Scientific Pierce Luciferase reporter genes are human codon optimized and offered in a plasmid vector that is optimized for mammalian expression. The Gaussia,Gaussia-Dura (a mutant form of the Gaussia luciferase gene that confers better bioluminescent signal stability than native luciferase) and Cypridina luciferase proteins are secreted into the cell culture media, allowing for live cell monitoring of reporter activity. The light signal produced by these three secreted luciferases is considerably greater than the signal from either native Firefly or Renilla luciferases. Although greater than 85% of the Gaussia, Gaussia-Dura and Cypridina luciferase protein is secreted, the remaining intracellular portion can be robustly detected in lysate, facilitating multiplexing with other intracellular luciferases.
The Green Renilla luciferase is an intracellular protein with a greater serum stability as well as higher light output than native Renilla and Firefly luciferases. Consequently, Green Renilla reporter assays are more sensitive than assays employing native Renillaluciferase.
The Red Firefly luciferase is an intracellular protein with an emission peak shifted into the red spectrum compared to the native Firefly luciferase, which allows multiplexing with Gaussia, Cypridina and Green Renilla luciferases.
Single- vs. Dual-spectral Reporter Assay Kits
Single-spectral Reporter Assay Kits
Thermo Scientific Pierce Luciferase Reporter Assay Kits for single luciferase assays are offered in both flash and glow formats.
Flash-type assays provide a short but intense burst of light for maximum sensitivity. Thus, the Flash assay reagents may require the use of a luminometer with reagent injectors for the greatest signal strength and reduced variability.
Glow-type assays provide a less-intense but longer-duration signal that is amenable to higher sample throughput and do not require reagent injectors.
Dual-spectral Reporter Assay Kits
Thermo Scientific Luciferase Dual Assay Kits are highly sensitive flash-type assays that can detect both a primary experimental luciferase reporter and a second luciferase reporter for normalization. The first luciferase signal reports specific experimental conditions, and the second signal reflects transfection efficiency and cell viability.
As shown in the first video below, dual-spectral luciferase reporter assays offer a one-step procedure for spectrally resolving the signals from two different luciferases. By adding two different substrates to the same assay reagent, two luciferase signals are measured simultaneously, and the distinct light signals are separated using filters. Thus, two different luciferases can be detected in a single sample without the need for a quenching step.
We offer three dual-spectral reporter assay kits, in which Gaussia, Cypridina or GreenRenilla is multiplexed with Red Firefly luciferase. Two of these kits include a secreted luciferase (Gaussia or Cypridina) along with the intracellular Red Firefly luciferase, while both luciferases in the third kit are not secreted (Renilla and Red Firefly).The second video below details how the dual-spectral luciferase kits with secreted luciferases can be used to perform time-course reporter assay, because secreted luciferase can be collected in the supernatant at various time points throughout the experiment.
Additional multiplexing can be achieved through co-transfection of multiple luciferases with different substrate requirements into the same cells. For example, reporter plasmid constructs containing Gaussia (or Green Renilla) can be co-transfected with Cypridinaand Red Firefly plasmids. In this case, individual luciferase signals can be detected using the corresponding single assay kits with the same sample divided into separate wells.
Luciferase Assay Video Animations
Tables of Luciferase Features and Kit Types
Thermo Scientific Luciferase reporter key features. Click links to navigate to product groups
in Flash Assay†
in Flash Assay
in Glow Assay
|†Flash assay reagents were added to sample wells containing either media or lysates obtained from HEK293 cells (24 hours post-transfection). Total light output (RLU) was captured by Thermo Scientific Varioskan luminometer (integration time = 0.1sec/reading).|
Thermo Scientific Luciferase compatibility chart for co-transfections. Click links to navigate to product groups.
|Thermo Scientific Dual-spectral Luciferase Reporter Assay Kits.