Fast relaxation imaging in living cells

Apratim Dhar, Martin Gruebele

Research output: Contribution to journalArticlepeer-review

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 lightemitting diode source, and a two-colorCCDcamera 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.

Original languageEnglish (US)
Article number28.1
JournalCurrent Protocols in Protein Science
Volume1
Issue numberSUPPL.65
DOIs
StatePublished - Aug 2011

Keywords

  • FRET
  • FReI
  • Fluorescence microscopy
  • GFP
  • Kinetics
  • Protein folding
  • Thermal denaturation

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry

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