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Firefly luciferase is a protein with a molecular weight of 61kDa . It is an ideal choice for reporter genes in the above applications because it has no background fluorescence in host cells, does not require post-translational modification, and is fast, reliable and convenient to detect. The Bio -Steady ^TM One-Step Firefly Luciferase Assay Kit is a one-step detection kit developed for firefly luciferase that is stable in luminescence and suitable for high-throughput screening . This kit uses D-luciferin as a substrate. In the presence of ATP, magnesium ions and oxygen, it can be catalyzed by firefly luciferase to undergo oxidative decarboxylation to produce activated oxyluciferin and generate bioluminescence (Figure 1). Bioluminescence is proportional to the promoter activity of luciferase, which indirectly reflects the biological activity of the cell stimulant.

Figure 1. Luminescence mechanism of firefly luciferase substrate

This kit can provide a signal half-life of more than 5 hours in commonly used cell culture media and does not require sample pretreatment. Under standard laboratory conditions , a throughput rate of thousands of samples per hour can be achieved with high reproducibility, making it very suitable for high-throughput and ultra-high-throughput (96/384/1536-well) detection of firefly luciferase reporter gene expression in mammalian cells. It can detect as low as 10-20 ^mol of luciferase, and has a good linear relationship in the enzyme concentration range of 10-20 to 10-14 mol ^. The ^{comparison of} luminescence value decay of different luciferase detection kits is shown in Figure 2.

Note: The actual readings may vary due to differences in cell number, transfection effect, detection instrument, etc. The data in the figure are for reference only.