The luciferase assay system uses a lysate to gently and rapidly extract luciferase from eukaryotic cells and use its substrate to detect luciferase activity. The detection steps are as follows:
1. Add the lysis buffer to lyse the transfected cells.
2. Transfer the above lysate into a microplate or tube (select the type of equipment used according to the test).
3. Add all the enzyme reaction components (must include the substrate fluorescein) to start the chemiluminescence reaction.
4. Detect the emitted fluorescence using a fluorometer or a liquid scintillation counter.
Experimental steps:
Note: This procedure is commonly used to detect luciferase expression in eukaryotic cells transfected with the firefly luciferase gene and is not suitable for detection of bacterial luciferase.
1. On the first day of the experiment, the cells were digested and inoculated (select appropriate cells according to specific experiments) and cultured in a 35 mm cell culture dish in a 37 ° C incubator at 5% CO 2 and saturated humidity overnight.
2. When the cell density reaches 70%, the Luciferase reporter plasmid, LacZ expression plasmid and other plasmids (according to specific experiments) are co-transfected with cells (transfection methods are selected according to specific experiments, such as calcium phosphate precipitation method, PEI, lipofectamine2000, etc.) can).
3. After 24-36 h of transfection, the culture was aspirated and the cells were washed with pre-cooled PBS (no calcium and magnesium ions).
Note: The enzymatic reaction of luciferase is inhibited by trace amounts of calcium, so cells transfected with calcium phosphate should be washed thoroughly to remove the calcium-containing medium before collecting the cells.
4. 350 μl of pre-cooled harvest buffer was added to each dish and lysed at 4 ° C or ice for 10 min.
5. During cell lysis, a sufficient amount of 1.5 ml microcentrifuge tubes was prepared, and ATP buffer and luciferin buffer were mixed at a ratio of 1:3.6 to form a reaction solution, and 100 μl per tube was dispensed.
6. An equal volume of cell lysate (100 μl) was sequentially taken to the centrifuge tube in step 5, mixed rapidly, and the absorbance was read on a Luminometer.
Note: The luminescence reaction will decay rapidly. The absorbance value must be read within 5 s after adding the cell lysate to the reaction solution.
7. Be sure to read the absorbance of all samples in the same way.
8. The remaining lysate was assayed for activity of LacZ and its reading was used as an internal standard to correct luciferase readings.
9. Analyze the data by plotting the corrected readings.
Note: Fluorescein is easily oxidized, and unused fluorescein should be discarded.
Precautions:
1. The luciferase assay reagent should be pre-equilibrated at 15-25 °C before reacting, and then automatically or manually detected according to specific conditions. When using a liquid scintillation counter, mix gently after adding the luciferase assay reagent.
2. If the extract cannot be detected immediately after cell lysis, the sample can be stored on ice for about 5 hours and stored at -70 °C for several months. Do not freeze and thaw repeatedly to avoid a decrease in enzyme activity.
3. When the cold sample is detected by the above method, the signal intensity will decrease by 5-15%.
4. In the case of high luciferase activity, the signal is out of the linear range (signal overflow) and the sample can be diluted with the lysate.
5. Do not store diluted samples. If necessary, BSA should be added to the diluted sample to a final concentration of 2.5 mg/ml to maintain sample stability.
6. Some fluorometers require 1-2s of stability before performing the test. Therefore, it is recommended to perform the test operation after a period of time after turning on the power.
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