Application of photobleaching for measuring diffusion of prion proteins in cytosol of yeast cells

Measurement of fluorescence recovery after photobleaching (FRAP) is a non-invasive technique for studying protein dynamics in real time in living cells. FRAP studies are carried out on proteins tagged with green fluorescent protein (GFP) or one of its spectral variants. Illumination with high intensity laser light irreversibly bleaches the GFP fluorescence but has no effect on protein function. By photobleaching a limited region of the cytoplasm, the rate of fluorescence recovery provides a measure of the rate of protein diffusion. A detailed description of the FRAP technique is given, including its application to measuring the mobility of GFP-tagged Sup35p in [psi−] and [PSI+] cells.