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: Contribution to journalArticle

30 Citations (Scopus)

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)
Pages (from-to)345-363
Number of pages19
JournalMethods
Volume472
StatePublished - 2010
Externally publishedYes

Fingerprint

Stoichiometry
Luminance
Fluorescence
Cells
Proteins
Experiments
Titration
Fluorescence Spectrometry
Fluorophores
Calibration
Observation
Light
Spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Observing protein interactions and their stoichiometry in living cells by brightness analysis of fluorescence fluctuation experiments. / Chen, Yan; Johnson, Jolene; Macdonald, Patrick; Wu, Bin; Mueller, Joachim D.

In: Methods, Vol. 472, 2010, p. 345-363.

Research output: Contribution to journalArticle

Chen, Yan ; Johnson, Jolene ; Macdonald, Patrick ; Wu, Bin ; Mueller, Joachim D. / Observing protein interactions and their stoichiometry in living cells by brightness analysis of fluorescence fluctuation experiments. In: Methods. 2010 ; Vol. 472. pp. 345-363.
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