We express a variety of proteins in mammalian cells as fusion proteins with either enhanced green, enhanced yellow or enhanced cyan fluorescent protein (EGFP/EYFP/ECFP), all of which are chromatic variants of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria.

The localization of GFP-tagged proteins can easily be monitored in both live and fixed cells by fluorescence microscopy (see our Image Gallery for some examples). Filter sets can be obtained which distinguish between EYFP and ECFP, and between EYFP and EGFP, thus allowing dual-color imaging of two tagged proteins in the same cell (for review see Ellenberg et al., 1999).

In addition to providing information about intracellular localization of proteins, fluorescent tagging permits the analysis of protein dynamics in live cells. This is done via photobleaching approaches such as FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching). For review see White and Steltzer, 1999.

EYFP and ECFP also function as a donor-acceptor pair for fluorescence resonance energy transfer (FRET), in which excitation of the donor (cyan) molecule leads to emission from the acceptor (yellow) molecule, provided that the proteins are close enough for energy transfer to occur. FRET can therefore be used to monitor direct protein-protein interactions between EYFP and ECFP fusion proteins in living or fixed cells (for review see Pollock and Heim, 1999).

Note: For details about such techniques as establishing cell lines stably expressing GFP fusion proteins and performing FRET analyses, visit our Protocols page.

Ellenberg, J., Lippincott-Schwartz, J. and Presley, J.F. 1999. Dual-colour imaging with GFP variants. Trends Cell Biol. 9:52-56.
Pollok, B.A., and R. Heim. 1999. Using GFP in FRET-based applications. Trends Cell Biol. 9:57-60.
White, J. and Stelzer, E. 1999. Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol. 9:61-65.