Here, I present a list of those drawbacks I personally encountered during my bench-sessions. Hoping this will help you getting the most out of your reporter assay. Feel free to discuss and implement in the comments, thanks.
#1 - Matrix effect. Hemoglobin and cytochromes in general, absorb luciferase light thus quenching the real brightness. Try always several dilutions before relying on your data, or switch to a red luciferase. BTW, 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 - An enzyme without activity. When you buy restriction enzymes, you know how many Units you get. The same is for any polimerase. Luciferase is an enzyme. When you buy recombinant luciferase, no vendor declares its enzymatic activity (moles of substrate processed per unit of time), making it difficult to standardize your standard.
#3 - Poor spatial resolution. This is due to two concomitant factors:
- poor antibodies. I never found a scientist saying he is comfortable with current available anti-luciferase antibodies. (Thus, forget immuno-stainings, confocal imaging and flow cytometry with your luciferase-sample).
- microscopy devices. Bioluminescence microscopy requires a very very dark room compared to fluorescence microscopy. Some microscopes are available for such a technique, but are poorly widespread, thus it is likely that your facility will not grant you to observe your luciferase-cells at the microscope.
#5 - Half-life variability. Don't forget to measure 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 either in live cells or in cell lysates.
Do you know other limitations?
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Nice post. Other limitations of luciferases are: requirement of oxygen and of ATP. Although the need of ATP can be an advantage in many cases (only live, metabollicaly active cells will glow), it can also be a drawback if you want to image/detect live "dormant" cells.
I have had the opportunity to play a little bit with the Olimpus luminescence microscope and I think that this technique has lots of potential.
Btw, which half lives have you got for the firefly luciferase? 3-12 h seems quite long to me! Do you know how any papers that measure the half-life of the luciferase in E. coli?
Regards,
Nuria
Thanks for the comment Nuria, I don't have in mind any paper dealing with luciferase half-life and bacteria. What I know is that in bacteria the lux operon could be an appealing alternative because of its ability to synthesize itself the substrate luciferin. I read about firefly luciferases optimized to have very extended half-lives (12h and more) the rationale was to stack several plates in the luminometer for high-throughput screenings. Personally I prefer destabilized ones: clearly an advantage of luciferase assays is the possibility to extend the experiment over the time dimension, this is completely thwarted if the reporter accumulates!
hello
can lucifrase be internalized in the cells or enter the cell by any means?
hello
can lucifrase be internalized in the cells or enter the cell by any means?
ramy-hilal@hotmail.com
Hello Ramy, usually is the plasmid containing the gene coding for luciferase that is put into the cells by transfection. I'm not aware of studies reporting that luciferase protein enters directly into the cells. You can test it by incubating your cells in presence of recombinant luciferase (available from Promega). After extensive cell washing, you can lyse the cells, do a normal luciferase assay, and compare the results with cells treated with a non-bioluminescent protein like BSA.
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