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Nanobead-based single-molecule pulldown for single cells
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DOI：10.1016/j.heliyon.2023.e22306.png)
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摘要
En 中文
Investigation of cell-to-cell variability holds critical physiological and clinical implications. Thus, numerous new techniques have been developed for studying cell-to-cell variability, and these single-cell techniques can also be used to investigate rare cells. Moreover, for studying protein-protein interactions (PPIs) in single cells, several techniques have been developed based on the principle of the single-molecule pulldown (SiMPull) assay. However, the applicability of these single-cell SiMPull (sc-SiMPull) techniques is limited because of their high technical barrier and special re-quirements for target cells and molecules. Here, we report a highly innovative nanobead-based approach for sc-SiMPull that is based on our recently developed microbead-based, improved version of SiMPull for cell populations. In our sc-SiMPull method, single cells are captured in microwells and lysed in situ, after which commercially available, pre-surface-functionalized magnetic nanobeads are placed in the microwells to specifically capture proteins of interest together with their binding partners from cell extracts; subsequently, the PPIs are examined under a microscope at the single-molecule level. Relative to previously published methods, nanobead-based sc-SiMPull is considerably faster, easier to use, more reproducible, and more versatile for distinct cell types and protein molecules, and yet provides similar sensitivity and signal-to-background ratio. These crucial features should enable universal application of our method to the study of PPIs in single cells.
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HETEROGENEITY
DYNAMICS
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IF:
3.6
论文数:
3.8W
被引数:
10.5W
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