Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells
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 combinat...
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doaj-5cfb3541d7a5456688c0644e66e248fc2021-07-23T13:45:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-07-01227300730010.3390/ijms22147300Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living CellsLaura M. Nederveen-Schippers0Pragya Pathak1Ineke Keizer-Gunnink2Adrie H. Westphal3Peter J. M. van Haastert4Jan Willem Borst5Arjan Kortholt6Victor Skakun7Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The NetherlandsDepartment of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The NetherlandsDepartment of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The NetherlandsLaboratory of Biochemistry, Wageningen University & Research, 6708 WE Wageningen, The NetherlandsDepartment of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The NetherlandsLaboratory of Biochemistry, Wageningen University & Research, 6708 WE Wageningen, The NetherlandsDepartment of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The NetherlandsDepartment of Systems Analysis and Computer Simulation, Belarusian State University, 220030 Minsk, BelarusProtein 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.https://www.mdpi.com/1422-0067/22/14/7300brightness and diffusion global analysis<i>Dictyostelium discoideum</i>dimeric proteinGFPFK506 binding protein 12fluorescence correlation spectroscopy |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Laura M. Nederveen-Schippers Pragya Pathak Ineke Keizer-Gunnink Adrie H. Westphal Peter J. M. van Haastert Jan Willem Borst Arjan Kortholt Victor Skakun |
spellingShingle |
Laura M. Nederveen-Schippers Pragya Pathak Ineke Keizer-Gunnink Adrie H. Westphal Peter J. M. van Haastert Jan Willem Borst Arjan Kortholt Victor Skakun Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells International Journal of Molecular Sciences brightness and diffusion global analysis <i>Dictyostelium discoideum</i> dimeric protein GFP FK506 binding protein 12 fluorescence correlation spectroscopy |
author_facet |
Laura M. Nederveen-Schippers Pragya Pathak Ineke Keizer-Gunnink Adrie H. Westphal Peter J. M. van Haastert Jan Willem Borst Arjan Kortholt Victor Skakun |
author_sort |
Laura M. Nederveen-Schippers |
title |
Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells |
title_short |
Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells |
title_full |
Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells |
title_fullStr |
Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells |
title_full_unstemmed |
Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells |
title_sort |
combined fcs and pch analysis to quantify protein dimerization in living cells |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2021-07-01 |
description |
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. |
topic |
brightness and diffusion global analysis <i>Dictyostelium discoideum</i> dimeric protein GFP FK506 binding protein 12 fluorescence correlation spectroscopy |
url |
https://www.mdpi.com/1422-0067/22/14/7300 |
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