Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2

A number of photoactivatable GFP-like fluorescent proteins (PAFPs) have been reported whose fluorescence can be switched on or whose fluorescent state can be modified by relatively intense irradiation at a specific wavelength. The use of these proteins gives unique opportunities to photolabel and track fusion proteins in a living cell. Here, we provide a protocol for the primary visualization, photoactivation and tracking of two monomeric PAFPs recently developed in our lab. Both these proteins, PS-CFP2 and Dendra2, are fluorescent and can be visualized before photoactivation. Upon photoactivation, their excitation and emission spectra undergo a dramatic red shift. The brightness of their initial and photoconverted states, along with the high dynamic ranges of both proteins, make them an attractive tool for protein photolabeling. Excluding genetic constructs cloning, cell culturing and transfection, the whole protocol may take anywhere from 10 min to several hours, depending on motility of the protein being studied.