All tags cell and tissue imaging tools Choosing a fluorescent protein

Choosing a fluorescent protein

Everything you need to know to choose the best fluorescent protein for biological imaging.

Since the original green fluorescent protein (GFP) gene was cloned in 19921, there has been an explosion in the variety of fluorescent proteins (FPs) available. They can be fused to a protein in transgenic cells or animals, conjugated to an antibody, or even used as a substrate in enzymatic reactions.

Before selecting a fluorescent protein for any of these applications, there are a number of key considerations to keep in mind.


FP considerationFP consideration

C- vs N-terminal fusion

Try both if possible; C-terminal fusion proteins are generally better. Confirm localization with an antibody.

Excitation and emission

Ensure you have correct lasers to excite, and that emission peaks do not overlap.

Brightness

Select the brightest FP that also meets your experimental requirements.

Maturation

Select a short maturation for fast/time-sensitive events.

Bleaching

Choose an FP with high photostability for experiments with a long duration.

Environmental conditions

Ensure the pH, temperature, and oxygen are optimized for your FP.

Codon optimization

Check the FP codon sequence is optimized/suitable for your species – especially when using older plasmids.

Oligomerization

Generally, try to use a monomeric FP.


To view the properties of some of the most popular fluorescent proteins, take a look at our quick reference card.


C vs N: which terminal?

Whether you choose to fuse your FP to the C or N terminal of your protein of interest largely depends on the protein itself: how it folds and whether the terminus you choose has a functional requirement or not. For example, if the C-terminal is folded inside the protein, you’re unlikely to receive any FP signal; or if your protein is post-translationally cleaved at the terminal your FP is fused to, then your FP will be removed from your protein of interest.

If you have the resources or your experiment is novel, it might be best to clone both C- and N-terminally tagged constructs to determine the best option. One research group found that more C-terminal fusion proteins localize to the intended subcellular compartment than N-terminally tagged fusion proteins2. However, it is important to stress that while C-terminally tagged proteins tend to localize and behave as expected, this is not always possible to predict.

You should confirm fusion protein localization by using an antibody against the native protein. Immunofluorescence can be used to check that the fusion protein localizes correctly; an immunoblot will help to confirm the fusion protein is the correct size and expressed at the expected levels; and co-immunoprecipitation can help to assess how the fusion protein interacts with known substrates3.

Excitation, emission, and brightness

If you plan on using multiple FPs, it is important to choose FPs with distinct emission peaks – as well as excitation peaks that you can target with your available lasers. If the emission peaks overlap, it will be difficult, or possibly impossible, to differentiate them.  

You typically want the brightest FP within your available spectra in order to achieve a clear signal and overcome any potential background fluorescence. Brightness values are a product of the protein’s extinction coefficient and quantum yield. However, the resulting number can be difficult to interpret, and so an FP’s brightness relative to a well-defined FP like EGFP, is a common alternative measure.

Maturation and bleaching

Maturation defines how long it takes an FP to fold correctly, form the chromophore, and begin to fluoresce. For time-sensitive events in live cells, a short maturation time can be important. Superfolder GFP (sfGFP) for example, can fold in under 10 minutes, while mOrange2 can take over four hours.

Bleaching is a measure of photostability, ie how long after excitation the chromophore loses the ability to emit light. If you plan on conducting lengthy time-lapse experiments, consider an FP with a high photostability. T-sapphire has a bleaching half-life (t½; time for an initial emission rate of x photons/s to reduce to half) of 25 seconds, but EGFP is much more stable, with a bleaching t½ of 174 seconds4.

Environmental conditions

Like most proteins, FPs are affected by pH, temperature, and oxygen levels. Depending on the environment you plan to use your FP in, you may need to either adjust the conditions slightly or select a more appropriate FP.

The pH can affect excitation and emission peaks, and the majority of FPs are sensitive to acid. Some FPs can change fluorescent intensity upon pH changes (eg pHTomato). The pKa value is a good indicator of pH sensitivity: it shows the pH at which half of the chromophores are fluorescent.

Temperature and oxygen levels both affect maturation times: hypoxic conditions tend to delay maturation times, as do temperatures outside of the FP’s optimal range (eg EGFP has been optimized to work at 37oC). However, newer FPs like UnaG, a GFP isolated from the Japanese freshwater eel (Anguilla japonica), fluoresces even when oxygen levels are low5.

Codon optimization 

As most FPs are derived from jellyfish or coral proteins – rather than something like the mammalian cells and tissues you are likely to use them in – there can be an interspecies difference in the amino acid codons used. This can lead to poor FP expression and therefore low signal.

Fortunately, many of the newer versions of FPs have been codon-optimized to reflect mammalian cell preferences. In GFP for example, Jürgen Haas and colleagues improved the signal 40–120 fold by modifying the GFP codon sequence6.

If you’re using an older plasmid to generate your fusion proteins, it may not contain a modified FP sequence. Check to see if your FP sequence has been modified for use in a certain species.

Oligomerization

It’s important to determine whether your FP is a monomer or dimer (monomers are usually denoted by an “m” prefixing the protein name, eg mCherry), and whether or not this affects your experiment. Many of the early FPs were prone to form oligomers, and oligomerization can affect the biological function of the fusion protein. EGFP, for example, is a monomer that can form dimers when used in high enough concentrations, which can distort subcellular organelles7 or disrupt experiments like FRET8.

Truly monomeric FPs are recommended in the vast majority of cases.


For more information on FPs, visit our GFP resources page.

References

1. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992).

2. Palmer, E. & Freeman, T. Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays. Comp. Funct. Genomics 5, 342–353 (2004).

3. Snapp, E. L. Fluorescent Proteins: A Cell Biologist’s User Guide. Trends Cell Biol. 19, 649–655 (2009).

4. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

5. Kumagai, A. et al. A bilirubin-inducible fluorescent protein from eel muscle. Cell 153, 1602–1611 (2013).

6. Haas, J., Park, E.-C. & Seed, B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6, 315–324 (1996).

7. Snapp, E. L. et al. Formation of stacked ER cisternae by low affinity protein interactions. J. Cell Biol. 163, 257–269 (2003).

8. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–6 (2002).

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