Selective photolabeling of proteins using photoactivatable GFP

Methods. 2004 Apr;32(4):445-50. doi: 10.1016/j.ymeth.2003.10.006.

Abstract

Today's cell biologists rely on an assortment of advances in microscopy methods to study the inner workings of cells and tissues. Among these advances are fluorescent proteins which can be used to tag specifically and, in many cases, non-invasively proteins of interest within a living cell. Introduction of DNA encoding the fluorescently tagged protein of interest into a cell readily allows the visualization of the protein's localization and time-lapse imaging allows the movement of the structure or organelle to which the protein is localized to be observed. To monitor the movement of the protein within the population, researchers generally have to highlight a pool of molecules by perturbing the steady-state fluorescence. This perturbation has traditionally been performed by photobleaching the molecules within a selected region of the cell and monitoring the recovery of molecules into this region or the loss of molecules within other regions. Fluorescent proteins are now available, which allow a pool of molecules to be highlighted directly by photoactivation. Here, we discuss the technical aspects for using one of these recently developed photoactivatable fluorescent proteins, PA-GFP.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Lasers
  • Microscopy, Fluorescence / methods
  • Mutagenesis, Site-Directed / genetics
  • Photochemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Spectrometry, Fluorescence / methods
  • Spectrophotometry
  • Staining and Labeling / methods*

Substances

  • Recombinant Fusion Proteins