Analysis of protein dynamics with tandem fluorescent protein timers

Methods Mol Biol. 2014;1174:195-210. doi: 10.1007/978-1-4939-0944-5_13.


Fluorescent timers (FTs) are fluorescent proteins that change color with time. FTs can be used as tags to follow protein dynamics in living cells. Recently we described a novel class of FTs called tandem fluorescent protein timers (tFTs). Each tFT is a tandem fusion of two different conventional fluorescent proteins having distinct kinetics of fluorophore maturation. tFTs suitable for studying protein dynamics on different scales can be generated from a broad range of commonly used fluorescent proteins. Here we describe how to establish new tFTs and consider potential pitfalls. We detail a protocol for quantitative fluorescence microscopy imaging and analysis of intracellular protein dynamics with tFTs in the budding yeast Saccharomyces cerevisiae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Intracellular Space
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence* / methods
  • Protein Transport
  • Proteins / metabolism*
  • Recombinant Fusion Proteins*
  • Saccharomyces cerevisiae / metabolism


  • Luminescent Proteins
  • Proteins
  • Recombinant Fusion Proteins