Revealing protein dynamics by photobleaching techniques

Methods Mol Biol. 2004;284:287-306. doi: 10.1385/1-59259-816-1:287.

Abstract

Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photobleaching (FRAP) to study protein kinetics in vivo. During photobleaching, fluorochromes get destroyed irreversibly by repeated excitation with an intensive light source. When the photobleaching is applied to a restricted area or structure, recovery of fluorescence will be the result of active or passive diffusion from fluorescent molecules from unbleached surrounding areas. Fluorescence loss in photobleaching (FLIP) is a variant of FRAP where an area is bleached, and loss of fluorescence in surrounding areas is observed. FLIP can be used to study the dynamics of different pools of a protein or can show how a protein diffuses, or is transported through a cell or cellular structure. Here, we discuss these photobleaching fluorescent imaging techniques, illustrated with examples of these techniques applied to proteins of the Saccharomyces cerevisiae pheromone response MAPK pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / genetics
  • Fluorescence Recovery After Photobleaching / methods*
  • Green Fluorescent Proteins
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • MAP Kinase Signaling System*
  • Microscopy, Confocal
  • Mitogen-Activated Protein Kinases / genetics
  • Mitogen-Activated Protein Kinases / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins
  • FUS3 protein, S cerevisiae
  • Mitogen-Activated Protein Kinases