Measuring protein mobility by photobleaching GFP chimeras in living cells

Curr Protoc Cell Biol. 2003 Aug:Chapter 21:Unit 21.1. doi: 10.1002/0471143030.cb2101s19.

Abstract

This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo-induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.

MeSH terms

  • Animals
  • COS Cells
  • Cell Survival
  • Chlorocebus aethiops
  • Diffusion
  • Fluorescence Recovery After Photobleaching*
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • Organelles / metabolism
  • Photobleaching*
  • Protein Transport
  • Recombinant Fusion Proteins / metabolism*
  • Transfection

Substances

  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins