Determination of luciferase in mammalian cell extracts

Summary

The assay of luciferase in mammalian cell extracts can be used to (1) measure the amount of luciferase in animal tissues, and (2) measure other enzymatic activities such as CAT, β-galactosidase, and alkaline phosphatase.

Operation method

Experimental determination of luciferase in mammalian cell extracts

Principle

Fluoresceinase is an ideal reporter gene because mammalian cells do not contain endogenous fluoresceinase, which immediately generates functional fluorescein once transcription is complete, and also has the highest quantum efficiency of its photoproducts among all chemiluminescent reactions, with high detection sensitivity. The luciferase reporter gene is widely used for miRNA target gene validation. The principle of luciferase reporter gene lies in the fact that miRNA acts mainly by acting on the 3'UTR of the target gene, and the luciferase plasmid can be constructed by building the 3'UTR region of the target gene to the back of the reporter gene Luciferase in pGL3-basic vector. Then transfected into cells, the inhibitory effect of miRNA on the target gene can be quantitatively reflected by detecting the change of reporter gene expression (monitoring the change of luciferase activity) after comparing overexpression or interference of miRNA. Combined with targeted mutagenesis and other methods to further determine the site of action of miRNA and target gene 3'UTR.

Materials and Instruments

DNA transfected mammalian cultured cells
Cell lysis buffer Luciferase assay buffer Luciferin solution Phosphate buffer without calcium and magnesium salts
Fluorophotometer and photometer placement tubes Rubber rods

Move

1. 24-72 h after transfection, wash the cells 3 times with phosphate buffer solution (PBS) free of calcium and magnesium salts at room temperature. change the PBS solution gently because some mammalian cells (e.g., human fetal kidney 293 cells) are easily dislodged from the culture dish when they are blown vigorously. Typically, the time for maximum expression of the luciferase gene in transfected cells is 24-72 h. 2. Each 100 mm dish should be filled with the luciferase gene and the luciferase gene should be expressed in the transfected cells.

2. For each 100 mm dish of transfected cells, add lml of ice-cold lysis buffer. The buffer is gently swirled, and then the lysed cells are scraped off the dish with a rubber mallet. Transfer the cell lysates to a 1.5 ml centrifuge tube. 3.

3. Centrifuge the cell lysate at maximum speed for 5 min at 4°C. Carefully transfer the supernatant to another new 1.5 ml tube. 4.

4. Determine the protein concentration of the lysate using a rapid colorimetric method such as the Bradford assay. Before determining the protein concentration, dilute the TritonX-100 to less than or equal to 0.1% to prevent interference. In this step, the cell lysate can be divided into small portions and stored at -70°C. Although luciferase is unstable in lysis buffer when stored at or below -20°C (Brasier et al. 1989), the enzyme can be safely stored at 4°C in a buffer containing 15% (V/V) glycerol and 1% (m/V) bovine haematocrit.

5. Tap the wall of the tube containing the lysate to gently mix the lysate. At room temperature, add 5-200ul of cell lysate to each fluorophotometer tube containing 360ul of luciferase assay buffer. The tubes are placed into the fluorophotometer.

In addition to the luciferase activity that can be measured using the fluorophotometer, there is another method in the column alternative: measuring luciferase activity with a scintillation counter.

6. The assay begins with 200ul of luciferase solution added to the fluorescence photometer tube, and then the light output is measured over a 2-60s time period at room temperature. The optimal length of time to measure light output is determined empirically and is related to the type of transfected cells as well as the particular substrate and luciferase used.

7. Determine the relative number of light units produced by each tube and determine the linear range of the measurement. Use an appropriate amount of cell lysis protein so that the value it produces in subsequent measurements lies in the middle of the linear range. The amount of protein required will vary depending on the strength of the promoter under study and the transfection efficiency of each experiment. Luciferase activity in cell lysates can be expressed as a number of relative light units or milligrams.

Caveat

Luciferin is the generic term for a substrate that can be catalyzed to emit light by the enzyme luciferase. The commonly used luciferase gene comes from the firefly, which acts on a specific luciferin substrate. Other luciferase enzymes such as those from sea pansies or a certain marine bacterium use luciferin substrates with different chemical properties. It was determined that the luciferin substrate used in the preceding buffer did match the luciferase gene in the expression plasmid.

Common Problems

In order to minimize the influence of intrinsic variability factors, such as: the number of cultured cells, the efficiency of cell transfection and lysis on the accuracy of the experiment, the plasmid with the luciferase gene was used as a control plasmid to co-transfect the cells with the reporter gene plasmid, which provided an internal control of the transcriptional efficiency, so that the results of the test would not be interfered with by the changes in the experimental conditions. During the measurement process, a firefly fluorescence signal was first generated when the luciferase detection reagent was added, so that the firefly luciferase activity was measured first. After quantifying the firefly fluorescence intensity, the Stop&Glo Reagent reagent is then added to the same sample to quench the above reaction and simultaneously initiate the sea kidney luciferase reaction for a second measurement, referred to as a dual luciferase reporter gene assay.

Sourced from Guide to Molecular Cloning Experiments (3rd Edition), next volume

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