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...
Saved in:
| Main Authors: | , , |
|---|---|
| 其他作者: | |
| 格式: | Article |
| 語言: | English |
| 出版: |
2017
|
| 主題: | |
| 在線閱讀: | https://hdl.handle.net/10356/83957 http://hdl.handle.net/10220/42890 |
| 標簽: |
添加標簽
沒有標簽, 成為第一個標記此記錄!
|
| 總結: | 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. |
|---|