Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experiments

Yan Chen, Jolene Johnson, Patrick Macdonald, Bin Wu, Joachim D. Mueller

Research output: Chapter in Book/Report/Conference proceedingChapter

34 Scopus citations

Abstract

A single fluorescently labeled protein generates a short burst of light whenever it passes through a tiny observation volume created within a biological cell. The average amplitude of the burst is related to the stoichiometry of the fluorescently labeled protein complex. Fluorescence fluctuation spectroscopy quantifies the burst amplitude by introducing the brightness parameter. Brightness provides a spectroscopic marker for observing protein interactions and their stoichiometry directly inside cells. Not all fluorescent proteins are suitable for brightness experiments. Here we discuss how brightness properties of the fluorophore influence brightness measurements and how to identify a well-behaved fluorescent protein. Protein interactions and stoichiometry are determined from a brightness titration. Experimental details of brightness titration measurements are described together with the necessary calibration and control experiments.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages345-363
Number of pages19
DOIs
StatePublished - Jan 2010

Publication series

NameMethods in Enzymology
Volume472
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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