Luciferase is my favorite reporter gene. The luciferase assay is the most common one because of several advantages (just have a look at vendor's sites). Although rarely reported, there are also disadvantages, but they are not mentioned. Here, I present 5 luciferase shortcomings I personally encountered at the bench. Hoping this will help you to get the most out of your reporter assay. Feel free to discuss and share other luciferase limitations in the comments, thanks.
#1 - The matrix effect. Hemoglobin, cytochromes and other heme-containing proteins present in your cell lysate are known to absorb luciferase light thus quenching the real luciferase brightness. When measuring the lysate at the luminometer, always try several dilutions before relying on your RLU. When making dilutions, take care of the total protein content: if you dilute too much your sample, luciferase can be broken or lost attached to plastic walls: include BSA 1 mg/mL in your diluting buffer.
#2 - The only enzyme without declared activity. What are RLUs? Why should you be happy with Relative Light Units? When you buy restriction enzymes, you know how many Units you get. The same is for any polymerase. Luciferase is an enzyme. However, no vendor is disclosing the enzymatic activity of their luciferase. So the luminometer just gives you some relative value, making difficult to standardize your assay. Similarly to the standard BSA dilution in the Bradford Assay, if you know the luciferase enzymatic activity, you can include a standard dilution in your assays (i.e., with recombinant luciferase). By doing so, you will convert RLUs to actual Luciferase Units and your values will be standardized for each experiment, with any luminometer, and with all the other scientists around the globe.
#3 - Poor spatial resolution. This is due to two concomitant factors:
- Poor antibodies. I never found a scientist saying to be comfortable with any anti-luciferase antibody. (Thus, forget immuno-staining, confocal imaging, flow cytometry and immuno-precipitation with your luciferase-sample).
- Microscopy devices. Bioluminescence microscopy requires a very dark room compared to fluorescence microscopy. Some microscopes come with bioluminescence imaging options, but they are poorly widespread: thus, it is likely that your facility will not grant you to observe any luciferase-positive cell at the microscope. This is a pity, because with one bioluminescence microscope, you can make a great Science paper like this one by David Suter.
#4 - Buy luciferin at $10,000/Kg. Luciferase requires a costly substrate (D-luciferin for Firefly and the very unstable coelenterazine for Renilla and Gaussia). This is not a real problem if you measure cell lysates at the luminometer, but the price of your experiment is going to grow if you plan to express luciferase in a host (i.e., cell implantation, transgenesis, adenovirus).
#5 - Half-life variability. Don't forget to measure the luciferase stability in your system: the luciferase half-life varies from 3- to 12-14 hrs according to different authors as it strongly depends on the host cell and the gene construct: just few amino-acid substitutions can severely affect the dynamics of proteosomal degradation. As a brutal method to extimate my luciferase half-life, I stop protein synthesis with cycloheximide and measure luciferase decay at different times both in live cells and in my cell lysates.
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