Double luciferase reporter gene detection kit

Firefly & Renilla Assay Kit（Double luciferase reporter gene detection kit）To provide an effective means of detecting gene expression levels, in DLR detection, the activities of firefly luciferase and Renilla luciferase can be sequentially detected in a single sample. Firstly, Luciferin is used as a substrate to detect the activity of firefly luciferase. Then, substances that inhibit firefly luciferase catalysis are added, and Coelenterazine is added to detect the activity of marine luciferase, achieving dual luciferase reporter gene detection. Through the bioluminescence system of luciferase and its substrates, gene expression can be detected very sensitively and efficiently. Usually, the transcriptional regulatory elements or 5 'promoter regions of the genes of interest are cloned upstream of Luciferase, or the 3' - UTR region is cloned downstream of Luciferase to construct a reporter gene plasmid. Then, cells are transfected and treated with appropriate drugs to lyse the cells. The transcriptional regulatory effect of drug treatment on the target gene is determined by detecting the level of luciferase activity. Sea kidney luciferase is more commonly used as an internal reference for detecting transfection efficiency to eliminate differences in cell number and transfection efficiency.
Firefly luciferase is a protein with a molecular weight of approximately 61 kD. In the presence of ATP, magnesium ions, and oxygen, it can catalyze the production of oxyluciferin from luciferin. During the oxidation of luciferin, a light signal is generated. Sea kidney luciferase is a protein with a molecular weight of approximately 36 kD. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to produce a light signal during coelenteramide oxidation. The light signal of this reagent kit can be measured using a chemiluminescence analyzer, enzyme-linked immunosorbent assay (ELISA) reader, or liquid scintillation counter. This kit has the characteristics of rapid detection, high sensitivity, wide detection range, and no interference from endogenous cellular activity.
Product Features:
Fast: Cell lysis is completed within 10-15 minutes.
Convenience: The reagents are easy to prepare and the sample detection steps are simple.
Sensitivity: able to detect the lowest 10-20Mol of luciferase.
The linear range of enzyme concentration can reach 8 orders of magnitude.
Product components:

1. Cell lysis
(1) Remove the culture medium and gently wash twice with PBS (adherent cells can be directly subjected to this operation, suspended cells need to be collected by centrifugation). Add 1 x Lysis Buffer according to the following scheme (dilute component A 4:1 with sterile water), then place the culture plate on a micro shaker and shake at room temperature for 15 minutes to fully lyse the cells.
Note: Cracking products can be stored at room temperature for 6 hours and can be stored for a long time at -70 ℃ (cracking products cannot be repeatedly frozen and thawed).
(2) Centrifuge the lysate at 10000-15000 rpm for 3-5 minutes, and transfer the supernatant into a new EP tube for subsequent testing.
Note: After cell lysis, it can be detected immediately or frozen for further testing when needed. Frozen samples need to be thawed to room temperature before testing.
2. Preparation of working fluid
(1) Restore all components to room temperature.
(2) Dissolve component C thoroughly with component B to prepare a 0.2 mg/mL firefly luciferase working solution.
Note: Firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount used in a single experiment is small, it is recommended to divide it into small sizes according to the amount used in a single experiment.
(3) Dilute component E with component D to prepare a working solution of marine luciferase. The dilution method is to add 1 ul of component E to 49 µ L of component D.
Note: Sea kidney luciferase working solution needs to be prepared and used immediately.
3. Chemiluminescence value detection
(1) Turn on the instrument with chemiluminescence detection function, such as a multifunctional enzyme-linked immunosorbent assay (ELISA) reader, according to the instrument operation manual. Set the parameters, the measurement time is 10 seconds, and the measurement interval is 2 seconds.
(2) When measuring each sample, take 20-100 ul of the sample (if the sample volume is sufficient, add 100 ul; if the sample volume is insufficient, reduce the amount appropriately, but the amount used in the detection well should be consistent). 1 × Lysis Buffer is a blank control.
(3) Add 100 ul of firefly luciferase detection solution and measure the RLU (relative light unit) value (it is recommended to set the shaking mixing function on the ELISA reader).
Note: As the luminescence is instantaneous, it is recommended to add firefly luciferase working solution and perform testing immediately.
(4) Add 100 ul of sea kidney luciferase working solution and measure the RLU (relative light unit) value (it is recommended to set the shaking mixing function on the ELISA reader).
(5) Using firefly luciferase as an internal reference, divide the RLU value obtained by firefly luciferase assay by the RLU value obtained by sea luciferase assay. Compare the activation levels of target reporter genes among different samples based on the obtained ratios. If firefly luciferase is used as an internal reference, similar calculations can also be performed.

-Store at 20 ℃. C component is recommended to be pre prepared with sterile water as a 2 mg/mL storage solution, while B component, D component, and C component prepared as a storage solution,
Perform small batch packaging according to experimental requirements. All testing working fluids are recommended to be prepared and used immediately to avoid repeated freezing and thawing.

Fluorescent enzyme reporter gene detection kit,Double luciferase reporter gene detection kit