The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex.
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.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
Hieng Chiong
HC
Tie
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
Viswanadh
V
Madugula
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore. Electronic address: lulei@ntu.edu.sg.
Lei
L
Lu
D015153:Blotting, Western; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D015778:Minor Histocompatibility Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D022022:Nuclear Pore / Q000737:chemistry* / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D059008:Time-Lapse Imaging / Q000379:methods; D014162:Transfection
D015778:Minor Histocompatibility Antigens; C440250:NUP133 protein, human; C104835:NUP88 protein, human; D028861:Nuclear Pore Complex Proteins; C508154:Nup93 protein, human; D049452:Green Fluorescent Proteins
Fluorescence microscopy; Nuclear pore complex; Nucleoporin; Single molecule; Stoichiometry