Dual luciferase assay is an everyday well assessed technique in my lab. I use a bicistronic plasmid carrying two ORFs coding for Renilla luciferase and Firefly luciferase, respectively, and consider their enzymatic activity as an indication of their quantity.
However, in some cases this system happens to be far from precise: you always deal with enzymatic activity values but, apart from using their ratio as a way to infer relative change between them, you can hardly consider the activity value as a measure of the real quantity of translated luciferase protein.
I would like to know about the real protein concentration of both luciferases.
Using luciferase-specific antibodies does not help, as Firefly and Renilla luciferases are targeted by two different antibodies with different affinities, so again one cannot compare them to measure the absolute quantity of translated luciferase protein.
Is there any known standard equivalence between a given luciferase activity value and the quantity of the respective luciferase protein?
Thank you in advance for any help.
It is depend from a lot factors. Bioluminescence of luciferase does not correspond to protein concentration directly. Luciferase activity needs exact definition sure.
I agree with Dr. Belobrov. If you take e.g. firefly luciferase, there are several types both wild-types and recombinant versions. For a given type the intensity of the light is proportional to the amount of enzyme but the proportionality factor varies between the different luciferases. It also varies with reaction conditions. Try to get hold of a preparation of the specific enzymes you use in your assays and store aliquots for each run of the assay. The firefly luciferase we use is stable for many years if stored at -18C. I've attached a paper on how to optimize assays based on firefly luciferase. Hopfully it may be of some interest.
Thank you very much for the information attached. What I actually need is to know the ratio between the amount of Firefly luciferase and Renilla luciferases after transfection of a bicistronic plasmid. For example, if I get a ratio of luciferase activities of about 0,2, I should know the proportionality factors linking each enzymatic activity to the amount of the respective enzyme in order to know if Renilla luciferase is really present at a concentration 5 times higher than Firefly luciferase or if this ratio concerns strictly their activity.
I'm not surprised about the fact that each different luciferase has its own proportionality factor. So, as far I understood, the only way to get this information is to perform assays at different concentrations to determine a proportionality factor.
Cannot I really infer anything about the amount of luciferase proteins from their activity?
If you use the two abs in excess to get saturation on antigens, you can use the immune signal. Prefer abs from the same animal to use the same secondary on blots,and detect them paralel. That is the simplest case, of course.
Hi Piero, I am totally confuse with your question of trying to measure the actual amount of the two lucieferase proteins.....????? Unless you are working on the "function" of those two protein I don't see the reason behind doing that...???? As far my understanding the Luciferase reporters are "a tool to measure the transcriptional activity"....... and then you correlate those luciferase activities with actual mRNA level and if possible then the protein made out of that "specific gene" regulated by that specific promoter, cloned upstream of the Luciferase reporter gene.... These luciferase protein themselves have nothing to do with the function of the promoter cloned upstream to those luciefrase genes....!!!!???? And even if say you are trying to measure these protein by using "an artificial system of bicistronic plasmid transfection in the cells..... which is btw miles away from PHYSIOLOGICAL "levels of expression" will give you no answer....!!!!! if you increase the plasmid concentration in transfection, then they will make more protein.... isn't it???? as it is well known fact that the transfected PLASMIDS are 100% artificial system.....!!!!! unless you somehow integrate the PROMOTER and Lucifer's gene construct in the whole GENOME the transcription and translation read out are still "artificial".... as you don't have any control over location of promoter reporter integration in the genome....!!! so the bottom line is " I am beside myself to think and suggest any reason why should you even try to measure these protein.... forget about HOW...????
Renilla and firefly luciferase are two completely different enzymes catalyzing different chemical reactions emitting light at different wavelengths. If you know the ratio between the light emission and the amount for each enzyme you must determine the proportionality factor between light emission and amount of enzyme for both of them. If you perform the assay in vivo this will be difficult. In vitro it is possible as described in previous message.
How about designing two sets of primers for firefly luciferase and Renilla luciferase respectively, then perform a RT-qPCR to determine the transcripts (mRNA) levels of the two genes?
That's a very good question for sure!
1) Luciferases are widely used as reporters (detected mostly by lytic assays or sometimes in real-time in live cells) - but at the same time usually no one knows actual luciferase concentrations and just relies on relative values. :(
As suggested earlier by Arne Lundin, the best is to get a hold of recombinant luciferase and do a calibration curve. You can determine the luciferase activity per cell and then calculate the concentration using the mean cell volume.
2) As mentioned by Hanzhong Ke above, how linear are the relations of [luc mRNA] - [luc protein] - [luc activity] is also an interesting question.
3) It would also be interesting to test the fusion of both luciferases and look if the activity ratio is the same at different expression levels.
Applying all mentioned above will give you a better understanding. Specifically the q PCR for firefly luciferase and Renilla luciferase genes and the luciferase activity using a proper calibration curve. All the best
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