Conditions for using FRAP as a quantitative technique--influence of the bleaching protocol.

Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent way of data analysis.