Single-cell western blotting

Abstract

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct 103 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.

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Figure 1: Single-cell western blotting.
Figure 2: scWestern blotting of NSCs.
Figure 3: scWesterns capture FGF-2 signaling dynamics.
Figure 4: scWesterns track NSC lineage commitment during differentiation.

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Acknowledgements

We acknowledge E. Connelly for assistance with cell culture and conventional western blots, K. Heydari at the UC Berkeley Cancer Research Laboratory Flow Cytometry Facility for assistance with interfacing with FACS; R. Lin, L. Bugaj, E. Woods and C. Nilson for critical discussion; and the QB3 Biomolecular Nanotechnology Center (BNC) at UC Berkeley for partial infrastructure support. A.J.H. was funded as a 2013 Siebel Scholar. D.P.S. was funded as a 2014 Siebel Scholar and by a California Institute for Regenerative Medicine training grant (TG2-01164). A.E.H. an Alfred P. Sloan Research Fellow in chemistry. This work was supported by a US National Institutes of Health (NIH) New Innovator Award (DP2OD007294 to A.E.H.), a Medical Research Program Grant from the W.M. Keck Foundation (to A.E.H.), a UC Berkeley Bakar Fellowship (to A.E.H.) and an NIH research project grant (R01ES020903 to D.V.S.).

Author information

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Authors

Contributions

A.J.H. and A.E.H. designed the scWestern. A.J.H., A.E.H., D.P.S. and D.V.S. designed experiments. A.J.H., Z.X. and C.-C.K. performed scWesterns and calibration experiments. D.P.S. performed NSC culture, stimulation and differentiation; conventional western blots; flow cytometry; and plate-based immunocytochemistry. A.J.H. and D.P.S. performed in-microwell ICC. A.J.H., D.P.S. and Z.X. performed confocal microscopy. Z.X. and A.J.H. designed software and performed analysis for cell/microwell scoring, immunoprobing quality control and fluorescence quantitation. Z.X. performed fluid dynamics simulations. Validation data were analyzed by A.J.H., D.P.S. and Z.X. All authors wrote the manuscript.

Corresponding authors

Correspondence to David V Schaffer or Amy E Herr.

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Competing interests

A.J.H., Z.X., C.-C.K. and A.E.H. are inventors on pending patents related to scWestern blot methods. A.E.H. holds equity interest in Zephyrus Biosciences.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–27, Supplementary Table 1 and Supplementary Notes 1–8 (PDF 13747 kb)

Supplementary Software

Software and readme files for automated cell/microwell counting and scWestern blot analysis. (ZIP 26 kb)

EGFP+ NSC lysis, protein separation, and immobilization

Inverted fluorescence microscopy video at 10x magnification showing EGFP+ NSC lysis, protein separation upon application of electric field (dark moving bands), and UV immobilization steps in a scWestern gel. Black frames in the video correspond to a period of UV exposure of the scWestern gel after protein separation. Note retention of EGFP in the scWestern gel at the end of the UV exposure step. Vertical extent of the field of view is approximately 700 microns. (MP4 1616 kb)

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Hughes, A., Spelke, D., Xu, Z. et al. Single-cell western blotting. Nat Methods 11, 749–755 (2014). https://doi.org/10.1038/nmeth.2992

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