title: Measuring protein stability in living zebrafish embryos using fluorescence decay after photoconversion (FDAP).
abstract: Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.
authors: Department of Molecular and Cellular Biology, Harvard University.
authors: Katherine W
authors: KW
authors: Rogers
authors: Systems Biology of Development Group, Friedrich Miescher Laboratory of the Max Planck Society.
authors: Alexander
authors: A
authors: Blässle
authors: Department of Molecular and Cellular Biology, Harvard University.
authors: Alexander F
authors: AF
authors: Schier
authors: Systems Biology of Development Group, Friedrich Miescher Laboratory of the Max Planck Society; patrick.mueller@tuebingen.mpg.de.
authors: Patrick
authors: P
authors: Müller
mesh_terms: D000818:Animals; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D055668:Photochemical Processes; D055550:Protein Stability; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000032:analysis* / Q000378:metabolism*
chemical_list: D029961:Zebrafish Proteins
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