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Nat Methods. 2014 Jul;11(7):749-55. doi: 10.1038/nmeth.2992. Epub 2014 Jun 1.

Single-cell western blotting.

Author information

1
1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] [4].
2
1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA. [4].
3
1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.
4
1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA. [4] Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA. [5] Helen Wills Neuroscience Institute, UC Berkeley, Berkeley, California, USA.
5
1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA.

Abstract

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.

PMID:
24880876
PMCID:
PMC4077215
DOI:
10.1038/nmeth.2992
[Indexed for MEDLINE]
Free PMC Article

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