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...

Full description

Saved in:
Bibliographic Details
Main Authors: Tie, Hieng Chiong, Madugula, Viswanadh, Lu, Lei
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2017
Subjects:
Online Access:https://hdl.handle.net/10356/83957
http://hdl.handle.net/10220/42890
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-83957
record_format dspace
spelling sg-ntu-dr.10356-839572023-02-28T17:04:08Z The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex Tie, Hieng Chiong Madugula, Viswanadh Lu, Lei School of Biological Sciences Stoichiometry Single molecule 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. MOE (Min. of Education, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version 2017-07-18T01:38:53Z 2019-12-06T15:35:20Z 2017-07-18T01:38:53Z 2019-12-06T15:35:20Z 2016 Journal Article Tie, H. C., Madugula, V., & Lu, L. (2016). The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex. Biochemical and Biophysical Research Communications, 478(4), 1694-1699. 0006-291X https://hdl.handle.net/10356/83957 http://hdl.handle.net/10220/42890 10.1016/j.bbrc.2016.09.005 en Biochemical and Biophysical Research Communications © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 6 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Stoichiometry
Single molecule
spellingShingle Stoichiometry
Single molecule
Tie, Hieng Chiong
Madugula, Viswanadh
Lu, Lei
The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
description 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.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Tie, Hieng Chiong
Madugula, Viswanadh
Lu, Lei
format Article
author Tie, Hieng Chiong
Madugula, Viswanadh
Lu, Lei
author_sort Tie, Hieng Chiong
title The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
title_short The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
title_full The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
title_fullStr The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
title_full_unstemmed The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
title_sort development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex
publishDate 2017
url https://hdl.handle.net/10356/83957
http://hdl.handle.net/10220/42890
_version_ 1759853008946987008