Luc cell labeling service

The luciferase reporter gene system is a report system that uses luciferin as the substrate to detect the luciferase activity of firefly, and the biological fluorescence released during the reaction between the enzyme and the substrate reflects the biological activity of the enzyme. Firefly luciferase gene is used as a reporter gene to indicate the gene expression under different treatment conditions.

Firefly luciferase, 61 KDa in size, is a single-subunit protein that can catalyze fluorescein

(luciferin) oxidation to produce oxyluciferin;

The strongest luminescence wavelength of luciferin catalyzed by firefly luciferase is 560 nm.

Usually, the 5 ´ UTR or promoter of the target gene is cloned to the upstream of Firefly Luciferase, or the 3 ´ UTR or expected RNAi target sequence is cloned to the downstream of Firefly Luciferase, and the transcriptional regulation of the promoter or regulatory element is detected by detecting the reaction activity of firefly luciferase with the substrate. Renilla Luciferase was used as an internal reference to eliminate the differences between experimental groups such as cell number or transfection efficiency.

1. Cell planking, cell density during transfection: generally speaking, when the cell density reaches 60%~80%, transfection can achieve high transfection efficiency. However, the optimal transfection density of different cells is not the same. Therefore, it is suggested that the optimal transfection density of a certain cell can be confirmed first through pre-experiment.

2. Transfection According to the efficiency evaluation, expand the system to carry out the corresponding transfection example: 12-well plate, system 500 μ L

3. Lying cells

After the transfection to the corresponding time, discard the culture medium, wash the PBS once, completely cover the cells with the cell lysate, and fully mix it. Add the cell lysate in the following way to fully lyse the cells, about 30 min~1 h

For reference:

4. Fluorescence detection

1) Take 100 μ L cell lysate, added to the enzyme label plate. According to the needs of the experiment, 3 holes and 5 holes can be set for repetition;

2) Join 5 μ L Firefly luciferase reaction solution, shake the plate and mix it evenly to detect the activity of firefly luciferase. The detection should be completed within 30 minutes as far as possible.

5) Analyze data. Note: Because the expression of enzyme varies in different cells, the amount of substrate varies according to different cells, and the optimal range can be referred to 5-20 μ L。