.png){kind=icon}
返回
Single-molecule pull-down for studying protein interactions
.png){kind=icon}
OA
AI
DOI：10.1038/nprot.2011.452.png)
.png){kind=icon}
原文求助.png){kind=icon}
分享.png){kind=icon}
收藏
摘要
En 中文
This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.
Keyword:
COMPLEXES
CELLS
FRET
期刊
IF:
16
论文数:
4.0K
被引数:
5.6W
机构
引用论文
Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules荧光辅助分子分选: 通过单个分子的交替激光激发分析结构和相互作用
OAAI
OAAI分享收藏Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution.png)
ANALYTICAL CHEMISTRY
ANALYTICAL CHEMISTRYIF6.7
PREAI
分享收藏
查看更多内容