It is a question of experimental variation. If it were possible to measure a 4.5% difference in fluorescence with a precision of, say, +/-1%, then 4.5% would be sufficient.

It is a question of experimental variation. If it were possible to measure a 4.5% difference in fluorescence with a precision of, say, +/-1%, then 4.5% would be sufficient.

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Thank you adam, very useful.

I have to admit that i'm not very strong in the statistics, so can you specify better what do you mean saying precision? DO you mean between cells in the same experiment, or between different experiments? which test would you suggest, Standard error or SEM?

I wasn't being specific about the term precision. You have to be able to tell whether the difference you observe between treatments is statistically significant to whatever level of confidence you require (P-value). This can be done using the average and standard deviation (or standard error) of multiple repeated measurements, and a t-test.

I don't know the details of your experiment, but I would guess that you will make measurements of multiple cells for each treatment in each experiment and calculate the average value of the individual measurements. You would then use the standard deviations to perform the t-test, where n is the number of individual measurements. If you do the experiment multiple times and take the average of the experimental averages, then you should use the standard error, where n is the number of experiments.

It might also be helpful to do a few different positive controls as well just to get a better feel for the range of FRET efficiencies for your system.