In this review you will find 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 1992, there has been an explosion in the variety of fluorescent proteins (PF) available. They can be fused with 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
To see the properties of some of the most popular fluorescent proteins, take a look at Abcam’s Quick Reference Card.
C VS N: WHAT TERMINAL?
If you choose to fuse your PF to the C or N terminus of your protein of interest, the choice of which end to use will largely depend on the protein itself. You should evaluate how it folds and whether the end you choose has a functional requirement or not. For example, if the C-terminus is folded into the protein, it is unlikely to receive any signal from the PF; Or if your protein is post-translationally cleaved at the end to which its PF is fused, then your PF will be removed from your protein of interest.
If you have the resources or if your experiment is novel, it might be best to clone both ends with terminal C and N tags to determine the best option. A research group found that more C-terminal fusion proteins are located in the predicted subcellular compartment than N-terminal fusion proteins. However, it is important to emphasize that while C-terminal labeled proteins tend to localize and behave as expected, this is not always possible to predict.
You must confirm the location of the fusion protein using an antibody against the native protein. Immunofluorescence can be used to verify that the fusion protein is correctly located; an immunoblot will help you confirm that the fusion protein is the correct size and is expressing at expected levels; and coinmunoprecipitation can help evaluate how the fusion protein interacts with known substrates.
EXCITATION, EMISSION AND BRIGHTNESS
If you plan to use multiple PFs, it is important to choose a PF with different 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.
Generally, the brightest PF within your available spectra is desired to achieve a clear signal and overcome any possible background fluorescence. The brightness values are a product of the extinction coefficient of the protein and the quantum yield. However, the resulting number can be difficult to interpret, so the brightness of a PF relative to a well-defined PF as EGFP is a common alternative measurement.
RIPENING AND WHITENING
Maturation defines how long it takes to properly fold a PF, form the chromophore, and begin to fluoresce. For time-sensitive events in living cells, a short maturation time may be important. Superfolder GFP (sfGFP), for example, can fold in less than 10 minutes, while mOrange2 can take more than four hours.
Whitening is a measure of photostability, that is, how long after excitation the chromophore loses its ability to emit light. If you plan to conduct long time-lapse experiments, consider a FP with high photostability. Sapphire T has a bleaching half-life (t½; time for an initial emission rate of x photons / s to be halved) of 25 seconds, but EGFP is much more stable, with a bleaching t½ of 174 seconds .
Like most proteins, FPs are affected by pH, temperature, and oxygen levels. Depending on the environment in which you plan to use your PF, you may need to slightly adjust the conditions or select a more appropriate PF.
PH can affect excitation and emission peaks, and most FPs are acid sensitive. Some FPs can change fluorescent intensity with changes in pH. 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 affect maturation times: hypoxic conditions tend to delay maturation times, as do temperatures outside the optimal PF range (eg EGFP has been optimized to work at 37 ° C) . However, newer FPs such as UnaG, a GFP isolated from the Japanese freshwater eel (Anguilla japonica), fluoresces even when oxygen levels are low.
Since most FPs are derived from jellyfish or coral proteins, rather than from mammalian cells and / or tissues where you are likely to use them, there may be a difference between species in the amino acid codons used. This can lead to poor PF expression and therefore low signal.
Fortunately, many of the newer versions of the PF have been codon optimized to reflect the preferences of mammalian cells. In GFP, for example, Jürgen Haas and colleagues improved the signal 40-120 times by modifying the GFP codon sequence. If you are using an older plasmid to generate your fusion proteins, it may not contain a modified sequence for PF. Verify if your PF sequence has been modified to use on a specific species.
It is important to determine if your PF is a monomer or dimer (monomers are usually denoted by an “m” that precedes the name of the protein, eg mCherry), and whether or not this affects your experiment. Many of the early FPs were prone to oligomer formation, 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 organelles or interrupt experiments.