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Author Tie, Hieng Chiong ♦ Madugula, Viswanadh ♦ Lu, Lei
Source United States Department of Energy Office of Scientific and Technical Information
Content type Text
Language English
Subject Keyword APPLIED LIFE SCIENCES ♦ BIOMEDICAL RADIOGRAPHY ♦ COMPLEXES ♦ FLUORESCENCE ♦ MOLECULES ♦ POLYETHYLENE GLYCOLS ♦ STOICHIOMETRY
Abstract We report here an image-based method to quantify the stoichiometry of diffraction-limited sub-cellular protein complexes in vivo under spinning disk confocal microscopy. A GFP single molecule fluorescence standard was first established by immobilizing His-tagged GFP molecules onto the glass surface via nickel nitrilotriacetic acid functionalized polyethylene glycol. When endogenous nucleoporins were knocked down and replaced by the exogenously expressed and knockdown-resistant GFP-nucleoporins, the stoichiometry of the nucleoporin was estimated by the ratio of its fluorescence intensity to that of the GFP single molecules. Our measured stoichiometry of Nup35, Nup93, Nup133 and Nup88 is 23, 18, 14 and 9 and there are possibly16 copies of Nup107-160 complex per nuclear pore complex. - Highlights: • The method for preparing and imaging a GFP single molecule standard is developed. • The replacement of endogenous nucleoporins with exogenous GFP-tagged ones. • The estimation of the stoichiometry of nucleoporins in the nuclear pore complex.
ISSN 0006291X
Educational Use Research
Learning Resource Type Article
Publisher Date 2016-09-30
Publisher Place United States
Journal Biochemical and Biophysical Research Communications
Volume Number 478
Issue Number 4


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