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The Ultimate Guide to Choosing a Fluorescent Protein

image of fluorescent lights to represent choosing a fluorescent protein

Whether you’re just getting started working with fluorescent proteins and need help choosing one or are already a seasoned pro, this article gives some insight into developments in the field and how to go about choosing a fluorescent protein for your work.

You’re probably familiar with green fluorescent protein (GFP), which has had a long history in biotechnology in applications ranging from in vitro protein labeling to broader work investigating the structural properties and expression of cellular proteins. GFP and several of its variants (e.g., EGFP, sfGFP, muGFP) are readily found in nearly every biochemistry lab and are a go-to due to their popularity and availability.

With that said, there are now hundreds of different tailored fluorescent proteins that you can choose from that can improve your experiments and projects. Not only are there options with very specific excitation and emission wavelengths, but new and diverse proteins exist that reduce toxicity and maximize brightness across the visible spectrum.

In short, exploring the vast plethora of options in the fluorescent protein world is likely to improve your results as well as make you a better researcher!

6 Considerations When choosing a Fluorescent Protein

1. Brightness

Fluorescent proteins exhibit a range of brightness because of their individual properties and structure. While choosing the brightest protein in your chosen emission range seems like a no-brainer, you’ll want to keep in mind that theoretical brightness does not equate to “practical” brightness in your experimental setup.

The actual brightness of your protein may vary due to folding differences (based on what it is attached to) or the cell line you use. Consider testing multiple proteins that meet your experimental needs and establish a ‘baseline’ with a control condition.

2. Stability

As noted above, if the fluorescent protein you choose does not fold correctly, it may not fluoresce at all! The structural integrity of the fluorophore may be altered or compromised depending on where and how it is linked.

You may want to try linking your fluorescent protein at the N- or C-terminus of your protein of interest to determine the best linkage. While C-terminally-tagged proteins are most common and behave as expected more often than N-terminally tagged proteins,[1] this is not always the case.

Other factors such as the cell line and pH can impact fluorescent protein expression and fluorescence.

Luckily, directed evolution efforts have led to “enhanced” and “ultra-stable” fluorescent proteins that last longer in cell lines with higher temperatures and broader pH ranges. Some can even remain active in cell line workflows that involve chemically harsh tissue clearing methods.

3. Photostability

Photobleaching is a property of all fluorophores that results in the irreversible destruction of the fluorescent properties of the molecule. This includes fluorescent proteins! For live-cell imaging or other experiments with long time lapses, consider a fluorescent protein with high photostability. The longer the bleaching half-life (or the time for an initial emission rate of x photons per second to reduce to half), the more photostable the fluorescent protein.

4. Toxicity

Some fluorescent proteins have toxic effects on specific cell lines. Though not fully understood, cellular damage resulting from fluorescent protein expression can result from reactive oxygen species generation, initiation of apoptosis, damage by immune mechanisms—not to mention the tetramerization and aggregation behaviors of proteins.

Take a good look at the proteins you consider and their effects on cells by reviewing the literature before diving too far into an experiment.

5. Experimental setup

Your experimental limitations and expectations are arguably the most critical factors in deciding which fluorescent protein to use.

For instance, your experiment may require not one but multiple fluorescent proteins that interact (like in FRET to determine interactions of two targets). Perhaps you are running an experiment that requires very little autofluorescence, or has specific kinetic or fluorescence turnover needs.

Just as important, you’ll need to verify that your protein of choice is compatible with your microscope optics and filter sets. Look to your colleagues, core facility staff and the literature to identify what your needs are and choose a fluorescent protein based on these factors.

6. Cost and availability

Luckily, the genetic sequences of the most commonly used fluorescent proteins are readily available and in a diverse array of vectors (more on this below!). Obtaining and using fluorescent proteins is cheaper and easier than ever!

Why Consider a New Fluorescent Protein?

Biochemists and biologists have been working relentlessly for decades to engineer new and improved fluorescent proteins to help resolve the issues listed above.

Often, a few changes at key locations in the protein dramatically improve stability, brightness, and photostability. Even if you feel ‘settled’ in using a particular protein, you should periodically review if it is the best choice for your work.

One such example is with GFP. Consider the many varieties that exist beyond the wild-type today! Improvements are ongoing for a diverse palette of fluorescent proteins. Since science is always marching forward, don’t hesitate to explore alternatives to those you or your lab have not traditionally used.

For instance, developments in the field of “split” fluorescent proteins, in which fragments of a protein reassemble to form fully functional versions, have led to self-complementing epitope-tagging systems.

This technology allows researchers to add relatively small peptide tags rather than whole proteins, allowing for greater versatility and stability. For example, the “SunTag” sequence resulted in as many as 24 GFPs associating with a target protein!

Another example is the many new variants of mCherry engineered with red-shifted fluorescence and reduced cytotoxicity.

Some Popular Fluorescent Proteins

Below are some of the most commonly used and well-known fluorescent proteins, including their in vivo structure. Keep in mind that this is only a small selection of the wide breadth of those available today! For a more comprehensive table including even more proteins and details, check out this primer to fluorescent proteins at MicroscopyU.com.

Fluorescent ProteinExcitation
Min
(nm)

Emission
Max
(nm)

Relative
Brightness
(% of EGFP)

Quantum YieldMolar
Extinction
Coefficient (M-1cm-1)

in vivo
Structure

Green Fluorescent Proteins
GFP (wt)395/475509480.7721,000Monomer*
eGFP4845071000.6056,000Monomer*
Superfolder GFP4855101600.6583,300Monomer*
T-Sapphire399511790.6044,000Monomer*
Blue Fluorescent Proteins
EBFP2383448530.5632,000Monomer*
mTagBFP399456980.6352,000Monomer
Cyan Fluorescent Proteins
mTurquoise434474750.8430,000Monomer*
CyPet435477530.5135,000Monomer*
mTFP1 (Teal)4624921620.8564,000Monomer
Yellow Fluorescent Proteins
Topaz5145271690.6094,500Monomer*
mCitrine5165291740.7677,000Monomer
YPet5175302380.77104,000Monomer*
ZsYellow1529539250.4220,200Tetramer
mBanana540553130.76,000Monomer
Orange Fluorescent Proteins
Kusabira Orange25515651180.6263,800Monomer
mOrange5485621460.6971,000Monomer
dTomato5545811420.6969,000Dimer
TagRFP5555841420.48100,000Monomer
DsRed5585831760.7975,000Tetramer
mTangerine568585340.3038,000Monomer
Red Fluorescent Proteins
mRuby5586051170.35112,000Monomer
mApple5685921090.4975,000Monomer
mStrawberry574596780.2990,000Monomer
mCherry587610470.2272,000Monomer
mRaspberry598625380.1586,000Monomer
mPlum590649120.1041,000Monomer
* Weak Dimer

Resources to Help You Choose (and Use!) the Right Protein

Luckily, fluorescent proteins are widely available and used in labs throughout the world. Gone are the days when receiving a plasmid bearing a protein-producing gene of your choice (or worse yet, having to clone one yourself) was a multi-month ordeal. Consider the resources below for selecting and procuring fluorescent proteins.

Published Literature

You can find helpful resources in the published literature from looking at what fluorescent proteins others have used in your field or technique, keeping up to date with new fluorescent proteins that become available, or through reviews on the topic. [2,3]

Your Lab/Collaborators

If your lab or surrounding labs and collaborators already use fluorescent proteins, they may be able to provide a good recommendation for your application. They may even provide a suitable fluorescent protein. Using existing local resources is often the most convenient and cost-effective option.

FPbase

FPbase is an online database that provides pertinent fluorescent protein information, including sequences and evolutionary timelines, for over 750 proteins- and they are constantly growing as users add entries! [4]

Their interactive spectra viewer and table with details such as oligomerization type, protein size, and brightness are especially useful for anyone looking to do a comprehensive search. Each entry includes the original protein it was derived from, allowing you to see how multiple versions of a single protein are related.

Olympus Color Palette

The popular microscopy company Olympus also has a reasonably detailed list of proteins organized by emission wavelength and includes a brief history of several variants. Importantly, Olympus provides recommendations on filter types and further details pertinent to any cell microscopy user. While not an exhaustive list of all variants, it is an excellent starting point for those new to using biological fluorophores.

Plasmid Repositories

Once you’ve decided on using a new, cutting-edge fluorescent protein for your work, you may need to obtain the genetic sequence in a vector for transformation or transfection.

Addgene, one of the most well-known repositories, has a massive collection, including plasmids.

They have conveniently organized a collection of plasmids harboring fluorescent protein genes and categorized them by use. You can also custom order genetic sequences from companies like Genewiz or Twist Bioscience and insert them into the vectors of your choice.

Bitesize Bio

Of course, Bitesize Bio has a wealth of articles that review the basics of fluorophores and troubleshooting techniques. From PALM imaging to automated microscopy, we’ve got you covered. And don’t forget to check out The Microscopists podcast episode featuring Rita Strack, who engineered several improved variants of the red FP DsRed!

Do you have any favorite fluorescent proteins for unique applications, or have you “upgraded” one in your lab to get better results? Or are you new to using them and want to learn more? Let us know in the comments below!

References

  1. Palmer E & Freeman T. (2004) Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays. Comp Funct Genomics. 5(4):342-53.
  2. Shaner N et al. (2005) A guide to choosing fluorescent proteins. Nature Methods 2:905-909.
  3. Lu K, Vu CQ, et al. (2019) Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells. Int J Mol Sci. 20(22):5784.
  4. Lambert T. (2019) FPbase: a community-editable fluorescent protein database. Nature Methods 16:277-278.

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