Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging

• Autor(a) principal: Bodor, Dani L.
• Data de Publicação: 2012
• Outros Autores: Rodríguez, Mariluz Gómez, Moreno, Nuno, Jansen, Lars E.T.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.7/614
• topic: Animals; Cell Line; Cell Survival; Centromere; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Proteins; Recombinant Fusion Proteins

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.