title: Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP).
abstract: Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this unit, we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser-scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Day
authors.forename: Lewis J
authors.initials: LJ
authors.lastname: Kraft
authors.forename: Minchul
authors.initials: M
authors.lastname: Kang
authors.forename: Anne K
authors.initials: AK
authors.lastname: Kenworthy
mesh_terms: D000818:Animals; D019556:COS Cells; D003585:Cell Biology; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000378:metabolism; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods*; D015233:Models, Statistical; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D012984:Software
chemical_list: D018076:DNA, Complementary; D008055:Lipids; D011506:Proteins