Dual Luciferase Reporter Assay Kit

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SKU:
MORV0192
€143

Description

Product Information

The Dual Luciferase Reporter Assay Kit is used to detect gene regulation by transfecting cells with a reporter plasmid and measuring the fluorescence intensity of Luciferin substrate to reflect the level of Luciferase expression. To achieve dual luciferase reporter gene detection, firefly luciferase is detected with Luciferin as a substrate, and renilla luciferase is detected with coelenterazine as a substrate, while inhibiting the activity of Firefly luciferase. Dual Luciferase Reporter Assay Kit can detect the expression of Luciferase regulated by gene elements sensitively and efficiently. Usually, the transcriptional regulatory element is cloned upstream of Firefly luciferase, or the 3'- UTR regulatory region is cloned downstream of firefly luciferase. The transfected cells are induced by corresponding stimulator and lysed to determine the luciferase activity. The stimulatoryinducing effect of the regulatory elements is evaluated by luciferase activity. Renilla luciferase acts as an internal reference for correcting transfection efficiency to eliminate differences in cell number and transfection efficiency between wells. Firefly luciferase catalyzes the emission of Luciferin at 560 nm, and renilla luciferase catalyzes the emission of coelenterazine at 465 nm.

Product Components

Components MORV0192- 100 rxns

5 x Cell lysis Buffer

10 ml

Reaction Buffer II (Luciferase)

10 ml

Luciferase Substrate (Lyophilized)

1 vial

Stop & Reaction Buffer

10 ml

Renilla Substrate

200 µl

Storage

Store at -20℃;
Dissolved and dispensed Luciferase Substrate can be stored at -70℃ for long term, or at -20℃ for less than one month.

Preparation and Guidelines

Additional materials required
PBS, pipette or multichannel pipette, immunoassay microplates (black is preferred), luminometer detector or full-spectrum microplate reader.

  • User Guides
    When used for the first time, the Reaction Buffer II (Luciferase) should be poured into a brown dark bottle containing lyophilized Luciferase substrate. Mix thoroughly and dispense according to the needs of use and keep away from light at -70℃.
    • Mix 5 × Cell Lysis Buffer and ddH2O in a ratio of 1 : 4 before use, and place on ice for use;
    • Renilla Substrate is dissolved in ethanol. For the first time, please centrifuge briefly. Carefully measure the volume of the solution in the tube. If the volume of the liquid is significantly reduced, please add ethanol to make up for the volume.
  • Place Renilla Substrate on ice for use. Calculate the actual usage, mix the appropriate amount of Stop & Reaction Buffer and Renilla Substrate in a ratio of 50 : 1, and keep it from light at room temperature;
  • The enzymatic reaction is sensitive to temperature, so the cell lysis solution and the detection substrate solution should be equilibrated to room temperature before the sample is applied for testing;
  • Detection instrument selection: Instruments capable of detecting chemiluminescence are suitable for this kit. But for the same sample, the value of background signal and measurement of different detection instruments may be different, so it is necessary to detect the background signal of fluorescent substrate in the pre-experiment. The values of the different instruments should not be compared horizontally. If a full-spectrum microplate reader is used for detection, it is recommended to use an opaque microplate and guarantee a certain interval between the detection wells.

Protocol

1. Cell Lysis
Discard the cell culture medium and wash the cells twice with PBS. Add the appropriate amount of 1 × Cell Lysis Buffer as recommended in the table below. Stand still or shake for 5 min at room temperature, pipette up and down and transfer the cell lysate into a 1.5 ml centrifuge tube. Centrifuge for 2 min, 12000 × g at room temperature, and collect the supernatant for subsequent testing

Cell Culture Plate 1 x Cell Lysis Buffer

6-well

500 µl

12- well

200 µl

24 -well

100 µl

48-well

50 µl

96-well

20 µl

If the expression level of luciferase is too low, the amount of Cell Lysis Buffer can be appropriately reduced to increase the protein concentration.

2. Firefly luciferase activity detection
Add 100 μl of Luciferase Substrate (which has been equilibrated to room temperature) to the detection tube or microplate. Carefully pipet 20 μl of the cell lysate into the test tube or the plate. Immediately after mixing rapidly, detect the Firefly luciferase reporter gene activity by
a luminometer detector or a full-spectrum microplate reader.


3. Renilla luciferase activity detection
Add 100 μl of freshly prepared Renilla Substrate solution to the above reaction solution, and immediately after mixing rapidly, detect the renilla luciferase reporter gene activity by a luminometer detector or a full-spectrum microplate reader.

Notes:

1. The optimum lysis time may vary for different cell lines. It is recommended to start from 5 min, and the lysis time can be extended to 10 min for a complete lysis. After the lysis is completed, please do not pipet the cells for a long time to prevent the production of large amounts of foam, which may affect the enzyme activity.
2. If the expression level of luciferase is too low, the amount of Cell Lysis Buffer can be appropriately reduced to increase the protein concentration. And increase the number of duplicate wells to reduce the difference between pores caused by low concentration expression to ensure the reliability of the results.
3. In general, the addition of the Stop & Reaction Buffer can inhibit the more than 99% of the activity of Firefly luciferase, however, there may be trace activity left. Therefore, it is recommended to control the RLU value of expression of renilla luciferase at a level comparable to or slightly higher than that of Firefly luciferase during transfection.
4. The fluorescence intensity is stable within about one minute after the lysate is in contact with the substrate. When using a single-tube chemiluminometer, to obtain the best results, the time interval between the mixing of different samples and substrates and the detection on the machine should be as consistent as possible. When using a full-spectrum microplate reader, the cell lysate should be added in the well first, then the detection substrate should be added and tested on the instrument as soon as possible. The measurement time can be set between 1 - 10 sec according to the intensity of the
fluorescence value. Increasing the detection time will increase the fluorescence reading of the sample and the background at the same time.

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