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Updated: Feb 14, 2026

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy
Megan J Kaliszewski1, Xiaojun Shi1, Yixuan Hou2
1Department of Chemistry, University of Akron, Akron, OH 44325, USA.
Abstract:
Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (fc) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of fc values which can be used to determine the oligomer state of membrane proteins from live-cell experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.
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