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Journal Article
Single-cell western blotting
2014
Overview
A single-cell Western (scWestern) blotting technique allows quantitative measurements of up to 11 protein targets from ~2,000 individual cells in under 4 hours, expanding single-cell heterogeneity studies to the proteome.
To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10
3
concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-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 (∼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.
Publisher
Nature Publishing Group US,Nature Publishing Group
