Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
Nano Lett. 2015 May 13;15(5):3610-5. doi: 10.1021/acs.nanolett.5b01153. Epub 2015 Apr 27.
We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.
我们开发了基于微图案化功能化表面结构的 GFP 标记蛋白复合物的原位单细胞下拉（SiCPull）。在这些支持物上培养的细胞用温和的去污剂裂解，并用全内反射荧光显微镜原位探测表面捕获的蛋白复合物。通过 SiCPull，我们通过监测从表面的解离来定量绘制各种信号转导和转录激活因子复合物的寿命，并在单分子水平上定义它们的计量比。
