Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging - PubMed (original) (raw)
Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging
Dani L Bodor et al. Curr Protoc Cell Biol. 2012 Jun.
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.
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