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

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.

FCT doctoral fellowships: (SFRH/BD/74284/2010, SFRH/BD/33567/2008); FCT grants: (BIA-BCM/100557/2008, BIA-PRO/100537/2008); Fundação Calouste Gulbenkian; European Commission FP7 programme; EMBO installation grant.