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Which fluorescent protein should I use?

Many fluorescent proteins (FPs) have been developed over the years, and which FP to use in an experiment depends on many factors.

Single-color experiments

For single-color experiments, green FPs are the most common choice. EGFP is the most popular green FP and is a good choice for many single-color studies. However, other green FPs, such as TurboGFP (a.k.a. maxGFP), may be better choices for certain applications. For example, TurboGFP has many more advanced features compared to EGFP, such as brighter green fluorescence, faster maturation, and high pH- and photo-stability, making it ideal for experiments that would benefit from early signal detection and high sensitivity. If a red FP is preferred, mCherry is a great choice for most experiments due to its monomeric structure, good fluorescent properties and low toxicity. It is particularly suitable for protein tagging or when cells are sensitive to toxicity or protein aggregation that may occur in other red FPs. The brighter dTomato works well in situations where dimerization and some potential aggregation of the FP is acceptable. Another good red FP is DsRed_Express2, which is a minimally cytotoxic version of red FP.

Multicolor experiments

For multicolor experiments that simultaneously employ multiple fluorophores (including FPs and other dyes such as DAPI), researchers must carefully consider the spectral properties of the fluorophores to ensure that they are spectrally distinguishable based on the excitation and/or emission filters on the available microscopes, flow cytometers, or other hardware used in fluorescence detection. Basically, the detection hardware should be able to read out the fluorescence signal from each of the multiple fluorophores used in the experiment without interference from other fluorophores. This can be done by using excitation filter (or laser) to produce excitation light of the proper frequency that only excites the fluorophore of interest. It can also be done by using emission filter to only allow the emitted fluorescence light from the fluorophore of interest to enter the detector. For two colors, a standard green FP such as EGFP plus a standard red FP such as mCherry should work well on virtually all fluorescence microscopes and flow cytometers. For three colors, a blue, green and red FP combination (e.g. TagBFP + EGFP + mCherry) or a cyan, yellow and red combination (e.g. CyPet + YPet + mCherry) can work well, as these combinations are easily separable on most fluorescence microscopes or flow cytometers.

Fluorescence resonance energy transfer (FRET)

FPs are widely used in many fluorescence resonance energy transfer (FRET)-based applications. FRET is a physical process during which an excited donor chromophore molecule transfers energy to an acceptor chromophore through nonradioactive dipole-dipole coupling. In observation, FRET leads to a reduction of donor’s fluorescence and an increase of the acceptor’s emission. There are a few prerequisites for FRET: The donor and acceptor must be in close proximity (10-100 Å); The excitation spectrum of the acceptor must overlap with the emission spectrum of the donor; The donor’s and acceptor’s transition dipole orientations must be in parallel. FRET is distance-dependent and the efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor. It is therefore very sensitive to small changes in distance, making it a powerful tool in many applications, such as studying DNA or protein structure, and investigating molecular interactions. The cyan-yellow donor/acceptor pair, CyPet-YPet, is commonly used in FRET-based approaches due to its good dynamic range.

Our recommendations:

Single Color: EGFP, TurboGFP, mCherry, dTomato, or DsRed_Express2 (These FPs can be used with DAPI.)

Two-color: EGFP + mCherry or TurboGFP + mCherry (These pairs can be used with DAPI.)

Three-color: TagBFP + EGFP + mCherry or CyPet + YPet + mCherry (The second combination can be used with DAPI when proper filters are used.)

FRET-based applications: CyPet - YPet

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