Description
Duo-Glow Luciferase Assay Kit (BA0180) (BA0180)
The Duo-Glow Luciferase Assay Kit (SKU: BA0180) is a bioluminescent assay that allows the sequential measurement of the activity of two different luciferases, firefly (FFL) and Renilla (RL), in a single sample. Dual reporter systems improve the accuracy of reporter assays, since one reporter gene reflects the promoter of interest while the second serves as a normalising control. In this assay the firefly luciferase luminescence is measured first following addition of the FFL Reagent; the RL Reagent is then added to the same well, simultaneously quenching the firefly signal and initiating the Renilla reaction. The light produced by both reactions is conveniently measured on a luminometer. The assay is extremely sensitive, detecting as little as 2 fg of luciferase, and is especially suited to quantifying dual luciferase expression in recombinant cells or cell-free transcription/translation reactions. It is intended for research use only.
| Product Name: | Duo-Glow Luciferase Assay Kit (BA0180) |
| SKU: | BA0180 |
| Detection Method: | Bioluminescent (dual reporter, luminometer) |
| Detection Range: | Detection of as little as 2 fg luciferase |
| Sample Type: | Recombinant cells and cell-free transcription/translation reactions |
| Species Reactivity: | All |
| Assay Time: | Within 20 min |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
A bioluminescent dual-reporter assay for the sequential measurement of firefly and Renilla luciferase activity in a single sample. Firefly luminescence is measured first after adding the FFL Reagent; the RL Reagent then quenches the firefly signal while initiating the Renilla reaction. Both signals are read on a luminometer. The assay detects as little as 2 fg luciferase and provides dual luciferase readings within 20 minutes.
- High sensitivity and wide detection range; detects as little as 2 fg luciferase
- Compatible with routine laboratory and high-throughput formats and any luminometer
- Readily automated on high-throughput liquid-handling systems
- Fast and convenient three-step assay allowing dual luciferase detection within 20 minutes
- Gene regulation studies including gene expression level and promoter characterisation
- Modulation of gene expression by receptors, transcription factors and small molecules
- High-throughput screening for gene modulators in drug discovery
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reagent preparation: reconstitute the FFL and RL Reagents fresh in Assay Buffer, weighing out sufficient amounts (2 mg/mL for FFL Reagent and 35 mg/mL for RL Reagent) and gently shaking until fully dissolved (~10 min). |
| 2 | Sample preparation: remove media from cell cultures, wash cells with PBS, add enough Lysis Buffer to cover the monolayer (e.g. 50 uL per well of a 96-well plate) and gently shake on an orbital shaker at room temperature for 15 min; transfer lysates to tubes for storage or assay directly in opaque culture plates. |
| 3 | If not assaying directly, transfer lysate to an opaque white plate (50 uL for 96-well plates, 12.5 uL for 384-well plates). |
| 4 | Add reconstituted FFL Reagent to each well (100 uL for 96-well plates, 25 uL for 384-well plates). |
| 5 | Measure firefly luciferase luminescence on a luminometer (integration 1-5 sec is typically appropriate). |
| 6 | Add reconstituted RL Reagent to each well (100 uL for 96-well plates, 25 uL for 384-well plates) and gently tap to mix. |
| 7 | Measure Renilla luciferase luminescence on a luminometer. |
The light intensity (RLU) is directly proportional to the luciferase concentration. For each individual sample, the RLU of the experimental reporter should be normalised by the RLU of the control reporter.
| Component | Quantity | Storage |
| Lysis Buffer | 25 mL | -20C |
| FFL Reagent | 25 mg | -20C |
| Assay Buffer | 25 mL | -20C |
| RL Reagent | 400 mg | -20C |
