title: Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging.
abstract: Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.
authors.affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal.
authors.forename: Dani L
authors.initials: DL
authors.lastname: Bodor
authors.forename: Mariluz Gómez
authors.initials: MG
authors.lastname: Rodríguez
authors.forename: Nuno
authors.initials: N
authors.lastname: Moreno
authors.forename: Lars E T
authors.initials: LE
authors.lastname: Jansen
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D002503:Centromere / Q000378:metabolism / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D011993:Recombinant Fusion Proteins