Volume 8 Issue 1
Feb.  2022
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Yirong Yao, Wenjuan Wang, Chunlai Chen. Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)[J]. Biophysics Reports, 2022, 8(1): 29-41. doi: 10.52601/bpr.2022.210026
Citation: Yirong Yao, Wenjuan Wang, Chunlai Chen. Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)[J]. Biophysics Reports, 2022, 8(1): 29-41. doi: 10.52601/bpr.2022.210026

Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)

doi: 10.52601/bpr.2022.210026
Funds:  Development of this procedure was supported by grants from the National Natural Science Foundation of China (21922704, 21877069 and 22061160466 to CC, and 22007054 to WW), funds from Beijing Advanced Innovation Center for Structural Biology and Beijing Frontier Research Center for Biological Structure to CC.
More Information
  • Corresponding author: chunlai@mail.tsinghua.edu.cn
  • Received Date: 14 July 2021
  • Accepted Date: 13 September 2021
  • Available Online: 24 February 2022
  • Publish Date: 28 February 2022
  • Liquid–liquid phase separation (LLPS) causes the formation of membraneless condensates, which play important roles in diverse cellular processes. Currently, optical microscopy is the most commonly used method to visualize micron-scale phase-separated condensates. Because the optical spatial resolution is restricted by the diffraction limit (~200 nm), dynamic formation processes from individual biomolecules to micron-scale condensates are still mostly unknown. Herein, we provide a detailed protocol applying dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to detect and quantify condensates at the nanoscale, including their size, growth rate, molecular stoichiometry, and the binding affinity of client molecules within condensates. We expect that the quantitative dcFCCS method can be widely applied to investigate many other important phase separation systems.
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