Combined FCS and PCH analysis to quantify protein dimerization in living cells

Laura M. Nederveen-Schippers, Pragya Pathak, Ineke Keizer-Gunnink, Adrie H. Westphal, Peter J.M. van Haastert, Jan Willem Borst, Arjan Kortholt*, Victor Skakun*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Original languageEnglish
Article number7300
Number of pages19
JournalInternational Journal of Molecular Sciences
Issue number14
Publication statusPublished - 2-Jul-2021


  • Brightness and diffusion global analysis
  • Dictyostelium discoideum
  • Dimeric protein
  • FK506 binding protein 12
  • Fluorescence correlation spectroscopy
  • Fluorescence fluctuation spec-troscopy
  • GFP
  • Photon counting histogram

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