Choosing the Right Fluorescent Protein

In 1994 the green fluorescent protein cloned from Aequorea victoria became the first in a long line of genetically encoded labels. Since that time, the fluorescent protein palette has expanded to cover the entire visual spectrum. With so many color variations and options, which fluorescent protein (FP) is best for your research? Three key factors are among the most important to consider: brightness, photostability, and aggregation.

Brightness is the most obvious factor that most researchers consider when choosing an FP. In general, the brighter the FP, the better it will perform under almost all experimental conditions. When evaluating an FP’s brightness, make sure to look at the critical optical parameters — extinction coefficient and quantum yield. The product of these two values for different FPs can be used to directly compare their brightness. Brighter FPs will have lower detection limits (i.e. the concentration at which the FP becomes visible above autofluorescence of other cell components), and will allow imaging with lower excitation light intensity, minimizing the possibility of phototoxic effects.

Photostability has increasingly become a consideration when researchers choose fluorescent proteins. Many FPs, even if they are initially quite bright, will photobleach under continuous excitation during imaging. In order to perform long-term imaging experiments or to do quantitative analysis, an FP with high photostability should be the first choice. Unfortunately, methods for measuring and reporting photostability vary widely in the scientific literature, so be sure to understand how your FP’s photostability was measured before trying to make comparisons!

Aggregation (or oligomerization) has been one of the major issues tackled in the development of FPs. Many wild-type FPs form tetramers, which aggregate badly when expressed as fusion tags in cells. Engineered monomeric forms of many FPs are now available, and these monomeric FPs should always be used when making fusion constructs. For simple expression markers, however, oligomerization is not usually a major concern, and the brightest possible FP should be used in this case.

As with other research tools, doing your homework and reading the primary literature is always the best approach to choosing the right FP for your project!

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Thursday, August 9th, 2012 Fluorescent proteins

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