Unit

UNIT 28.1 Fast Relaxation Imaging in Living Cells

  1. Apratim Dhar,
  2. Martin Gruebele

Published Online: 1 AUG 2011

DOI: 10.1002/0471140864.ps2801s65

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Dhar, A. and Gruebele, M. 2011. Fast Relaxation Imaging in Living Cells. Current Protocols in Protein Science. 65:28.1:28.1.1–28.1.19.

Author Information

  1. University of Illinois, Urbana, Illinois

Publication History

  1. Published Online: 1 AUG 2011
  2. Published Print: AUG 2011

Abstract

This protocol describes the technique of Fast Relaxation Imaging (FReI) as applied to protein folding inside living cells. The required modifications of a fluorescence microscope by addition of a diode laser temperature jump source, a yellow/blue switchable light-emitting diode source, and a two-color CCD camera to collect movies of protein dynamics inside cells are discussed. A description of how proteins are labeled for imaging, how cells are prepared for imaging, and how imaging of kinetics inside cells with millisecond time resolution is obtained, along with the complementary in vitro experiments, is also provided. The ability to carry out comparative in vitro and “in-cell” measurements on the same setup allows for direct comparison of the features distinguishing cellular protein folding (or other biomolecular processes) from studies performed in dilute solution. Curr. Protoc. Protein Sci. 65:28.1.1-28.1.19. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • protein folding;
  • fluorescence microscopy;
  • FRET;
  • FReI;
  • GFP;
  • kinetics;
  • thermal denaturation