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J Biol Chem. 2019 Feb 9. pii: jbc.RA119.007685. doi: 10.1074/jbc.RA119.007685. [Epub ahead of print]

Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.

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RIKEN Quantitative Biology Center, Japan.
National Institutes of Natural Sciences, National Institute for Basic Biology, Japan.
Graduate School of Biostudies, Kyoto University, Japan.
Division of Quantitative Biology, National Institutes of Natural Sciences, National Institute for Basic Biology, Japan.


Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd). However, only a limited number of parameters have been measured experimentally in living cells. Here, we describe an approach for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy (FCCS). First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy (FCS) to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, FCCS analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor (EGF) stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.


RSK; dissociation constant; extracellular-signal-regulated kinase (ERK); fluorescence correlation spectroscopy (FCS); fluorescence cross-correlation spectroscopy (FCCS); live cell analysis; nuclear translocation; protein-protein interaction; single cell analysis

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