I am starting a bimolecular fluorescence complementation (BiFC) assay. In the same vector, there will be 'protein A' (A) fused to 1/2 mVenus in one cassette and in another cassette there will be 'protein B' (B) fused to the other half of mVenus, along with mCherry separated by a P2A site. A & B interact to produce an mVenus signal that is proportional to frequency of interaction and protein expression levels. The idea is that the mVenus complementation efficiency - the proportion of available A/B that bind - is calculated as a ratio of mVenus/mCherry fluorescent intensities. Additionally, BiFC signal is expressed as a ratio of mVenus/mCherry fluorescent intensities to normalize for variable expression. However, mCherry has lower extinction coefficient, lower quantum yield, and therefore lower fluorescent intensity per protein. So, if there were 100% fluorescence complementation and perfectly equal expression of all 3 proteins, then you would expect the mVenus/mCherry ratio to be >1 since mCherry is inherently less bright - even though there are equimolar amounts of protein.
Wouldn't this overestimate BiFC fluorescent protein complementation efficiency?
Yes, i think the best would be to have a positive control where you expect mCherry to mVenus in 1/1 ratio, ideally a construct that carries both fluorescent proteins, a single copy of each.
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