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Single-cell western blotting
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DOI：10.1038/NMETH.2992.png)
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To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct similar to 10(3) concurrent single-cell western blots (scWesterns) in similar to 4 h. A microscope slide supporting a 30-mu m-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (similar to 200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
Keyword:
POLYACRYLAMIDE GELS
PROGENITOR CELLS
PROTEIN
SENSITIVITY
EXPRESSION
CYTOMETRY
DYNAMICS
KINASE
NESTIN
NOISE
期刊
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32.1
论文数:
7.3K
被引数:
12.7W
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