Fluorescence recovery after photobleaching: application to nuclear proteins

Abstract

Fluorescence redistribution after photobleaching (FRAP) has received increasing attention ever since it was first introduced into cell biological research. The method was developed in the 1970s, when its biological application mainly focused on the mobility of fluorescently labelled constituents of the cell membrane. The development of confocal scanning microscopy in the 1980s facilitated accurate investigation of the behaviour of molecules in the inside of cells without specialised equipment. However, FRAP did not yet become as popular as it is today, probably because of the dedicated and time-consuming methodology required to purify and label proteins or other compounds and, moreover, to inject them into cells. The revolution created by the development of GFP-technology finally lead to a tremendous boost of FRAP applications in studying the behaviour of proteins in the living cells. Finally, the ongoing increase of speed and memory of personal computers allows computer modelling of FRAP experiments for analysis of complex 3-D FRAP data, and for the development of new FRAP assays. Here we discuss several variants of FRAP on the basis of its application to the investigation of the behaviour of proteins in the living cell nucleus.