Protocol for the Use of Luciferase Genes

The use of luciferase genes is a common practice in various scientific fields, including molecular biology, genetics, and biotechnology. Luciferase genes encode enzymes that produce bioluminescence, allowing researchers to study gene expression, protein interactions, and cellular processes.

The process begins with the luciferase binding with its substrate, luciferin, in the presence of oxygen and ATP (adenosine triphosphate). The luciferase then catalyzes the oxidation of luciferin, leading to the release of light in a reaction that is highly efficient and specific to the enzyme.

• Add 4 times the volume of water to 1 volume of 5 × cell lysate reagent, so that the final concentration becomes 1 ×. Balance 1 × reagent and allow luciferase to analyze the reagent at room temperature.
• Remove the medium and carefully wash the cells twice with PBS buffer, so that the cells cannot be lost.
• Cover cells with 1 × cell lysate reagent (minimum volume).
• Scrape off the cells attached to the medium and transfer the dissolved cells to a microcentrifuge tube at 12,000 × g for 5 seconds.
• The cell extract at 20 μl room temperature was mixed with 10 μl luciferase analytical reagents.
• Enzyme activity detection.

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• Frozen luciferase analysis reagents cannot be dissolved above 25°C, and the newly dissolved reagents must be mixed before use.
• The optimal reaction temperature of luciferase is room temperature, and the enzyme activity can exist stably for several hours at room temperature, but the fluorescence quantum reaction half-life is 5 minutes.
• For bacteria, mix 40 μl of bacteria with 50 μl of transfer solution, add 10 μl of 1 M K₂HPO₄, pH 7.8, and 20 mM of EDTA. Quickly freeze the mixture with dry ice, then balance the mixture to room temperature in a test tube in room temperature water. 300 μl freshly prepared cleavage mixture was added (1 volume of fresh lysate was added to 2 volumes of 2 × cell lysate reagent, 5 mg/ml BSA) and the cultured cells were placed at room temperature for 10 minutes. The final reagent concentration was 1 × cell lysate, 2.5 mg/ml BSA, and 1.25 mg/ml lysozyme.

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