title: Measuring protein mobility by photobleaching GFP chimeras in living cells.
abstract: This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo-induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.
authors.affiliation: NICHD, National Institutes of Health, Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Erik L
authors.initials: EL
authors.lastname: Snapp
authors.forename: Nihal
authors.initials: N
authors.lastname: Altan
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
mesh_terms: D000818:Animals; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D015388:Organelles / Q000378:metabolism; D038761:Photobleaching; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins