Analysis of Protein Kinetics Using Fluorescence Recovery After Photobleaching (FRAP).
Fluorescence recovery after photobleaching (FRAP) is a cutting-edge live-cell functional imaging technique that enables the exploration of protein dynamics in individual cells and thus permits the elucidation of protein mobility, function, and interactions at a single-cell level. During a typical FRAP experiment, fluorescent molecules in a defined region of interest within the cell are bleached by a short and powerful laser pulse, while the recovery of the fluorescence in the region is monitored over time by time-lapse microscopy. FRAP experimental setup and image acquisition involve a number of steps that need to be carefully executed to avoid technical artifacts. Equally important is the subsequent computational analysis of FRAP raw data, to derive quantitative information on protein diffusion and binding parameters. Here we present an integrated in vivo and in silico protocol for the analysis of protein kinetics using FRAP. We focus on the most commonly encountered challenges and technical or computational pitfalls and their troubleshooting so that valid and robust insight into protein dynamics within living cells is gained.
Laboratory of Biology, School of Medicine, University of Patras, GR26500 Rio, Patras, Greece.
Nickolaos Nikiforos
NN
Giakoumakis
Laboratory of Biology, School of Medicine, University of Patras, GR26500 Rio, Patras, Greece.|IBM Research Zurich, Säumerstrasse 4, CH-8803, Rüschlikon, Switzerland.
Maria Anna
MA
Rapsomaniki
Laboratory of Biology, School of Medicine, University of Patras, GR26500 Rio, Patras, Greece. lygerou@upatras.gr.
Zoi
Z
Lygerou
D000818:Animals; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D012984:Software; D013223:Statistics as Topic / Q000379:methods
D011506:Proteins
Artificial neural networks; Fluorescence recovery after photobleaching; Live-cell imaging; Parameter inference; Protein kinetics; Stochastic hybrid models; easyFRAP