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Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins

Meckel, Tobias and Semrau, Stefan and Schmidt, Thomas and Schaaf, Marcel J. M. (2011):
Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins.
16, In: Journal of Biomedical Optics, (7), pp. 076016. [Article]

Item Type: Article
Erschienen: 2011
Creators: Meckel, Tobias and Semrau, Stefan and Schmidt, Thomas and Schaaf, Marcel J. M.
Title: Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins
Language: English
Journal or Publication Title: Journal of Biomedical Optics
Volume: 16
Number: 7
Divisions: 10 Department of Biology
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10 Department of Biology > Plant Membrane Biophysics
10 Department of Biology > Membrane Dynamics
Date Deposited: 25 Jul 2011 12:00
Identification Number: doi:10.1117/1.3600002
Alternative Abstract:
Alternative abstract Language
The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.UNSPECIFIED
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