Unit

UNIT 8.8 Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging

  1. Dani L. Bodor,
  2. Mariluz Gómez Rodríguez,
  3. Nuno Moreno,
  4. Lars E.T. Jansen

Published Online: 1 JUN 2012

DOI: 10.1002/0471143030.cb0808s55

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

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.

Author Information

  1. Instituto Gulbenkian de Ciência, Oeiras, Portugal

Publication History

  1. Published Online: 1 JUN 2012
  2. Published Print: JUN 2012

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. Curr. Protoc. Cell Biol. 55:8.8.1-8.8.34. © 2012 by John Wiley & Sons, Inc.

Keywords:

  • SNAP;
  • pulse-chase;
  • quench-chase-pulse;
  • centromeres;
  • automated quantification;
  • protein dynamics;
  • protein turnover;
  • quantitative fluorescence