The Thermo Scientific Pierce Firefly One-Step Luc Glow Assay Kit measures firefly luciferase activity in mammalian cell culture using a single reagent-addition step, making it ideal for high-throughput screening (HTS) applications.
This glow-type luciferase assay kit is designed for use with cultured mammalian cells that are transfected with vectors expressing the appropriate firefly luciferase reporter enzyme. Several features make the kit especially suitable for luminometers without injectors and for high-throughput screening (HTS) applications. The convenient, one-step, homogenous protocol minimizes handling steps to support the use of automation. Simply combine the supplied substrate solution and assay buffer to make a single working solution, and then add it to microplate wells containing the transfected, treated cells. The stable signal output (glow) of this system provides flexibility with regard to incubation time. When used with Thermo Scientific Firefly Luc Plasmids, the kit provides a highly sensitive bioluminescent reporter assay system for the detection of promoter or pathway activity.
- Sensitive – highly sensitive detection of firefly luciferase activity
- Odorless – reagents have no foul or toxic odor, eliminating the need to work in a fume hood
- Convenient – one-step, mix-and-read protocol; no additional reagents required
- Stable – increased signal half-life and stability compared to flash assays
- Simple – does not require a luminometer with injectors
- Automation-friendly – amenable to high throughput assays
Assay buffer and substrate solution (100X)
Firefly luciferase and luminometer or other instrument capable of monitoring luminescence, such as Thermo Scientific Luminoskan Ascent and Varioskan Flash Microplate Readers.
- Promoter studies for analyzing cis- and trans-acting regulatory elements
- Drug screening
- siRNA and miRNA screening
- Multiplexed assays to study off-target effects
- Protein localization reporter assays
- Signal transduction pathway analysis
Firefly luciferase is a 62kDa protein produced in nature by several species of the Lampyridae family of beetles, which includes the genera Photinus and Luciola. Thermo Scientific Red Firefly Luciferase has an emission range of 560-700nm (λmax = 613nm). Bioluminescent signal from firefly luciferase originates from the oxidation of D-Luciferin. Light output captured using a luminometer can be correlated with the amount of firefly luciferase protein produced and used to determine the activity of the promoter driving firefly luciferase expression. Firefly luciferase bioluminescence provides researchers with a non-radioactive assay for measuring transcriptional activity of regulatory elements. Firefly luciferase reporter activity can be used to determine promoter activity, to study upstream pathway activity in the cell, or to determine effect of activators or inhibitors.
||Firefly luciferase reaction.
||Thermo Scientific Pierce Firefly Luc One-Step Glow Assay Kit reagents result in stable bioluminescent signal. Working reagent was added to sample wells HEK293 transfected with CMV-Red Firefly or competitor CMV-Luc plasmids. 24 hours later, plates were processed using the one-step protocol. Readings were taken every 2 minutes with a Varioskan Flash Multimode Reader (integration time of 1sec/reading).
||Thermo Scientific Pierce Firefly Luc One-Step Glow Assay Kit reagents show excellent signal linearity over a range of luciferase concentrations. Cell lysate from a CMV-Red Firefly expressing cell line was serially diluted with complete media. An equal volume of working reagent was added to each sample, and readings were taken at 10 minutes with a Varioskan Flash Multimode Reader (integration time of 1sec/reading).
Review of Luciferase Reporters
Luciferase Reporter Assay Selection Guide
Red Firefly Luciferase Reporter Assay Vectors
Red Firefly LentiLuc Packaging Kit and Lentiviral Control Particles
TurboFect in vitro Transfection Reagent
Firefly Luciferase Flash Assay Kit
alamarBlue Cell Viability Assay Reagent
Pierce LDH Cytotoxicity Assay Reagent