Fluorescent tags

​​Fluorescent tags, such as GFP, are useful tools in detecting proteins.

Download your in-depth PDF guide here. From determining the best application to identifying common issues, our guide will help you make the most of your bench time.

• ​​Introduction to fluorescent tags 
• Excitation, emission, and brightness
• Maturation and bleaching
• Environmental conditions
• Codon optimization 
• Oligomerization
• GFP and mCherry products

Introduction to fluorescent tags

Since the original green fluorescent protein (GFP) gene was cloned in 1992, there has been an explosion in the variety of fluorescent tags available [Figure 1]. A large advantage of fluorescent tags is that they are non-toxic and can, therefore, be used in live cells. Despite being large tags, they tend to be minimally disruptive to most proteins¹. 

Although GFP, with a molecular weight of 26.9 kDa, is one of the most widely used fluorescent tags, there are several important points to consider.

_{Figure 1: These graphs show the excitation and emission spectra for different fluorescent proteins. More detail can be found in Table 1 below.} 

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_{Table 1}

Excitation, emission, and brightness

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If you plan on using multiple fluorescent tags, it is important to choose one 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 fluorescent tag within your available spectra 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, therefore, a fluorescent tag’s brightness relative to a well-defined tag, like EGFP, is a common alternative measure.

Maturation and bleaching

Maturation defines how long it takes a fluorescent tag 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 mOrange 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 a tag 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 seconds.

Environmental conditions

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

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

Additionally, temperature and oxygen levels both affect maturation times: hypoxic conditions tend to delay maturation times, as do temperatures outside of the fluorescent tags optimal range (eg EGFP has been optimized to work at 37°C). However, newer fluorescent tags like UnaG, a GFP isolated from the Japanese freshwater eel (Anguilla japonica), fluoresces even when oxygen levels are low³.

Codon optimization

As most fluorescent tags 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 expression and therefore low signal.

Fortunately, many of the newer versions of fluorescent tags 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 sequence⁴.

If you’re using an older plasmid to generate your fusion proteins, it may not contain a modified fluorescent tag sequence. As such, always check to see if your sequence has been modified for use in a certain species.

Oligomerization

It’s important to determine whether your tag 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 fluorescent tags 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 organelles⁵ or disrupt experiments like FRET⁶. Truly monomeric FPs are recommended in the vast majority of cases.

GFP and mCherry products

References

1. Crivat, G. & Taraska, J. Imaging proteins inside cells with fluorescent tags. Trends in Biotechnology 30, 8-16 (2012).
2. Shaner, N., Steinbach, P. & Tsien, R. A guide to choosing fluorescent proteins. Nature Methods 2, 905-909 (2005).
3. Kumagai, A. et al. A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 153, 1602-1611 (2013).
4. Haas, J., Park, E. & Seed, B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Current Biology 6, 315-324 (1996).
5. Snapp, E. et al. Formation of stacked ER cisternae by low affinity protein interactions. Journal of Cell Biology 163, 257-269 (2003).
6. Zacharias, D. Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells. Science 296, 913-916 (2002).