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| Product Name | Dual Luciferase Reporter Gene Assay Kit |
| Catalog No. | RESK-0149 |
| Description | This kit enables sensitive dual luciferase reporter gene detection. Firefly luciferase activity is measured first using luciferin as the substrate; the firefly reaction is then quenched while Renilla luciferase activity is measured in the same sample using coelenterazine as the substrate. Renilla luciferase serves as an internal control to normalize variations in cell number and transfection efficiency. Key features: Stronger lysis capability: thoroughly lyses the vast majority of cell types. Stronger signal: enables accurate detection of weak promoter expression. Wider linear range: the linear detection range spans more than 8 orders of magnitude of enzyme concentration. |
| Intended Use | Simultaneous quantitative analysis of firefly and Renilla luciferase reporter gene activities for studying the transcriptional regulation of promoters or regulatory elements, with Renilla luciferase as an internal reference to correct for differences in cell number and transfection efficiency. |
| Principle / Technology | Firefly luciferase (approximately 61 kDa) catalyzes the oxidation of luciferin to oxyluciferin in the presence of ATP, magnesium ions, and oxygen, emitting bioluminescence at approximately 560 nm. Renilla luciferase (approximately 36 kDa) catalyzes the oxidation of coelenterazine to coelenteramide in the presence of oxygen, emitting bioluminescence at approximately 480 nm. Both signals can be measured with a luminometer. Typically, the 5' UTR or promoter of the gene of interest is cloned upstream of firefly luciferase, or the 3' UTR downstream, so that firefly luciferase levels reflect the transcriptional regulation of the promoter or regulatory element. The kit first detects firefly luciferase activity with luciferin, then quenches that reaction while detecting Renilla luciferase activity with coelenterazine. |
| Detection Method | Sequential bioluminescence detection: firefly luciferase signal is read first, quenched, and the Renilla luciferase signal is then read in the same well using a luminometer or luminescence-capable microplate reader. |
| Sample Type | Cultured mammalian cells / cell lysates. |
| Performance Range / Specifications | Linear detection range spanning more than 8 orders of magnitude of enzyme concentration. Complete lysis of most cell types. Accurate detection of weak promoter expression. Firefly luciferase signal shows almost no decay over 30 min of continuous kinetic monitoring, well exceeding the time required for routine instrument operation. Residual firefly signal after quenching is extremely low and essentially negligible, ensuring no interference with subsequent Renilla luciferase measurement. Signal intensity is essentially unaffected after 5 freeze-thaw cycles. |
| Sensitivity / LOD | High sensitivity for both firefly and Renilla luciferase detection. |
| Reaction Conditions / Protocol | Lyse cells with the provided lysis buffer. Add the firefly luciferase reaction working solution and measure the firefly signal; then add the Renilla luciferase reaction working solution, which simultaneously quenches the firefly signal and initiates the Renilla reaction, and measure the Renilla signal. Both reaction working solutions should be prepared fresh before use. Firefly and Renilla activities are measured sequentially in the same well. |
| Components / Formulation | Cell lysis buffer, firefly luciferase reaction buffer/substrate (Component B firefly luciferase buffer is essential for both firefly and Renilla detection), and Renilla luciferase substrate (coelenterazine-based) with quenching reagent. |
| Storage Conditions | Store at -20 °C. Firefly and Renilla luciferase reaction working solutions should be prepared fresh and must not undergo repeated freeze-thaw; aliquot and store at -20 °C or -80 °C. Store substrates protected from light at low temperature; coelenterazine is recommended to be stored at -80 °C. |
| Shelf Life | 1 year when stored at -20 °C. |
| Package Specifications | 100 rxns 1000 rxns 10 rxns |
| Molecular Weight | Firefly luciferase: approximately 61 kDa. Renilla luciferase: approximately 36 kDa. |
| Shipping Conditions | Shipped on dry ice. |
| Regulatory / Compliance | For research use only. Not for use in diagnostic procedures. |
| Compatibility | Compatible with luminometers and microplate readers equipped with a luminescence module; use white opaque microplates for plate-based detection. |
| Application Notes / Precautions | Cell lysates can be stored at -80 °C for up to 6 months or at -20 °C for up to 1 month. The two substrates do not require strict light protection during handling, but should be stored protected from light at low temperature; coelenterazine is recommended to be stored at -80 °C. For co-transfection of dual reporter vectors, optimize the ratio by pilot experiments (e.g., Renilla to firefly vector ratios of 1:10, 1:20, 1:50, 1:100); a ratio yielding firefly luminescence higher than Renilla luminescence is preferred. The detection order cannot be reversed: the firefly luciferase buffer (Component B) is essential for the optimal reaction environment of Renilla luciferase; even when only Renilla luciferase is measured, Component B must be added. If lysis buffer runs out, a substitute can be prepared by adding 0.3 mL Triton to 10 mL PBS. If BCA protein quantification of lysates shows high background, dilute the lysate 5-fold before measurement. There is no fixed normal range for firefly and Renilla signal values; the FLuc/RLuc ratio and the statistical significance between experimental and control groups are the key readouts. |
For research use only, not for clinical use.
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