Robust single-molecule approach for counting autofluorescent proteins

Laurent Cognet, Catherine Tardin, Marie-Laure Martin Négrier, Christelle Breillat, Françoise Coussen, Daniel Choquet, Brahim Lounis
J. Biomed. Opt.. 2008-01-01; 13(3): 031216
DOI: 10.1117/1.2940600

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1. J Biomed Opt. 2008 May-Jun;13(3):031216. doi: 10.1117/1.2940600.

Robust single-molecule approach for counting autofluorescent proteins.

Cognet L(1), Tardin C, Négrier ML, Breillat C, Coussen F, Choquet D, Lounis B.

Author information:
(1)Universite Bordeaux, Centre de Physique Moleculaire Optique et Hertzienne,
Centre National de la Recherche Scientifique, 351 Cours de la Liberation, 33405
Talence, France.

Using single-molecule microscopy, we present a method to quantify the number of
single autofluorescent proteins when they cannot be optically resolved. This
method relies on the measurement of the total intensity emitted by each aggregate
until it photobleaches. This strategy overcomes the inherent problem of blinking
of green fluorescent proteins. In the case of small protein aggregates, our
method permits us to describe the mean composition with a precision of one
protein. For aggregates containing a large number of proteins, it gives access to
the average number of proteins gathered and a signature of the inhomogeneity of
the aggregates’ population. We applied this methodology to the quantification of
small purified citrine multimers.

DOI: 10.1117/1.2940600
PMID: 18601540 [Indexed for MEDLINE]

Auteurs Bordeaux Neurocampus