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Valerică Raicu
Physics Department, University of Wisconsin-Milwaukee
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
Current methods for determining membrane protein association in cells are either very time consuming, require complicated procedures, or lack the sensitivity needed to assess the effect of concentration or ligand binding on the observed oligomerization. To overcome these limitations, we provide a detailed protocol for quantifying the relative abundance and stability of oligomers of differing sizes using two-dimensional fluorescence intensity fluctuation (2D-FIF) spectrometry. The approach can be implemented using a standard laser scanning fluorescence microscope along with custom written software for image analysis. This method may be applied to evaluate the extent of oligomerization as a function of receptor concentration and is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest.
Keywords
Fluorescence microscopy, Fluorescence fluctuation spectroscopy, G protein-coupled receptor, protein-protein interaction, oligomerizatioin, quaternary structure
V.R. is a co-founder of Aurora Spectral Technologies, LLC, which provided the OptiMiS detection head used to build the two-photon microscope employed for part of the measurements described in this study.
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Posted 11 Jul, 2019
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Valerică Raicu
Physics Department, University of Wisconsin-Milwaukee
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 11 Jul, 2019
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Current methods for determining membrane protein association in cells are either very time consuming, require complicated procedures, or lack the sensitivity needed to assess the effect of concentration or ligand binding on the observed oligomerization. To overcome these limitations, we provide a detailed protocol for quantifying the relative abundance and stability of oligomers of differing sizes using two-dimensional fluorescence intensity fluctuation (2D-FIF) spectrometry. The approach can be implemented using a standard laser scanning fluorescence microscope along with custom written software for image analysis. This method may be applied to evaluate the extent of oligomerization as a function of receptor concentration and is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest.
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