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

Bodor, Dani L.; Rodríguez, Mariluz Gómez; Moreno, Nuno; Jansen, Lars E.T.

http://hdl.handle.net/10400.7/614

Use this identifier to reference this record.

Name:	Description:	Size:	Format:
Bodor_CPCB2012_preprint.pdf	artigo principal	2.48 MB	Adobe PDF	Download

Send Feedback

Authors

Bodor, Dani L.

Rodríguez, Mariluz Gómez

Moreno, Nuno

Jansen, Lars E.T.

Abstract(s)

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.

Keywords

Animals Cell Line Cell Survival Centromere Fluorescent Dyes Humans Microscopy, Fluorescence Proteins Recombinant Fusion Proteins

URI

http://hdl.handle.net/10400.7/614

Citation

Bodor, D. L., Rodríguez, M. G., Moreno, N. and Jansen, L. E. 2012. Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging. Current Protocols in Cell Biology. 55:8.8:8.8.1–8.8.34.