title: Fluorescence recovery after photobleaching (FRAP) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons.
abstract: FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate. In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares. This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.
authors.affiliation: National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, USA.
authors.forename: Chan-Ying
authors.initials: CY
authors.lastname: Zheng
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Petralia
authors.forename: Ya-Xian
authors.initials: YX
authors.lastname: Wang
authors.forename: Bechara
authors.initials: B
authors.lastname: Kachar
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D049229:Dendritic Spines / Q000737:chemistry*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006624:Hippocampus / Q000737:chemistry* / Q000166:cytology; D018613:Microscopy, Confocal; D009474:Neurons / Q000737:chemistry* / Q000166:cytology; D051381:Rats; D014162:Transfection
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins