Brightness experiments

Patrick J. MacDonald, Jolene Johnson, Yan Chen, Joachim D. Mueller

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

Abstract

This chapter presents an overview of quantitative fluorescence brightness experiments with special emphasis on single-color measurements of protein homo-interactions inside living cells. We discuss practical considerations in the choice of the fluorescent labels and the calibration measurements necessary for quantitative interpretation of brightness experiments. Methods to identify and avoid common pitfalls, such as bleaching and saturation, are addressed. We examine the interpretation of brightness data with moment analysis. In particular, we focus on how to avoid or correct for undersampling, as well as how to characterize and adjust for photon detector effects. We conclude by describing brightness titration experiments which determine the binding curve and stoichiometry of a protein from apparent brightness data.

Original languageEnglish (US)
Title of host publicationFluorescence Spectroscopy and Microscopy
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages699-718
Number of pages20
ISBN (Print)9781627036481
DOIs
StatePublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1076
ISSN (Print)1064-3745

Keywords

  • Afterpulsing
  • Autocorrelation
  • Dead time
  • Fluctuation
  • Photobleaching
  • Photon count moments
  • Stoichiometry
  • Titration
  • Two-photon

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  • Cite this

    MacDonald, P. J., Johnson, J., Chen, Y., & Mueller, J. D. (2014). Brightness experiments. In Fluorescence Spectroscopy and Microscopy: Methods and Protocols (pp. 699-718). (Methods in Molecular Biology; Vol. 1076). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-649-8_32