Choosing a Fluorescent Protein

Making informed fluorescent protein (FP) choices will increase the quality and reproducibility of your science. Even if the FP you’re currently using is “working,” it’s worth reassessing your choice. New fluorescent proteins with improved properties are published every year.

If you switch to a brighter/more photostable FP:

• You will be able to use less excitation light and/or lower expression level of the FP construct. Excitation light and overexpression can both perturb the biology in your sample.
• You may be able to acquire longer timelapses with increased temporal resolution.
• Dimmer structures may become visible.

• Brightness: Amount of light emitted by an FP for a given amount of excitation light
• Photostability: Rate of photobleaching
• Maturation time: FPs must undergo a chemical reaction in the chromophore before they become fluorescent. This is called maturation, and the time it takes varies between FPs.
• pH sensitivity: Most fluorophores become less bright in acidic conditions, but some FPs perform better than others at low pH.
• Dimerization: Native FPs are usually dimers. The FPs we use have been mutated to decrease dimerization, but there is still variation in the amount of dimerization that occurs even with the mutations to increase monomericity.

Testing multiple FPs is crucial for several reasons:

• FPs may behave very differently in different sample types. [see C. elegans example]
• FPs may perturb the biology of your sample in unpredictable ways
• Different FPs may perturb the biology of your sample in different ways

First, generate a list of candidate FPs.

• Look for FPs that have high brightness and photostability and are monomeric. Good starting points include recent reviews of FPs like this one published in 2016, or the Fluorescent Protein Database: FPbase.org.  However, take all these numbers with a grain of salt; FPs may behave differently in your system.
• Use a spectraviewer to check compatibility with the filter sets on your microscope. If you work in the NIC, each microscope has its own spectraviewer on the equipment page.
• Once you have a list of candidates, go to the primary literature. Find the original paper in which each FP was published. Look for any unexpected features, and keep an eye out for properties that might be relevant to your experiments. For example, pay close attention to pKa if you will be expressing the FP in an acidic cellular compartment.

Now that you have a list of FPs, request them from the authors of the original publication. Many authors now choose to distribute their FPs on Addgene. Make sure to sequence all constructs you receive, especially if you get them from somewhere other than the lab that generated the FP.

Next, test each protein in your biological system, on your microscope! This book chapter contains a nice protocol for comparing relative brightness and photostability of an FP in your cell type of interest.

At this point, you will hopefully have multiple FPs that are performing well in your brightness and photostability tests, but there’s more to validate:

• Validate that your FP-tagged protein behaves normally. Compare expression level, localization, function, stability, and folding of your FP-tagged vs. untagged protein using technique that don’t require visualizing the FP. For example, compare localization of the FP-tagged and untagged constructs using immunofluorescence.
• Validate that expression of the FP-tagged construct doesn’t alter the biology of your sample more generally. For example, compare doubling time in cells with and without your FP-tagged construct.
• Compare N- and C-terminal fusions. They may behave very differently.

If more than one FP comes out of this testing looking good, perform your experiments in parallel with both!