Robust single-molecule approach for counting autofluorescent proteins.
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.
Cognet
Laurent
L
Universite Bordeaux, Centre de Physique Moleculaire Optique et Hertzienne, Centre National de la Recherche Scientifique, 351 Cours de la Liberation, 33405 Talence, France. lcognet@u-bordeaux1.fr
Tardin
Catherine
C
Négrier
Marie-Laure Martin
ML
Breillat
Christelle
C
Coussen
Françoise
F
Choquet
Daniel
D
Lounis
Brahim
B
D000465:Algorithms; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000379:methods*
D049452:Green Fluorescent Proteins