Single-cell western blotting

• Published in: Nature methods Vol. 11; no. 7; pp. 749 - 755
• Main Authors: Hughes, Alex J, Spelke, Dawn P, Xu, Zhuchen, Kang, Chi-Chih, Schaffer, David V, Herr, Amy E
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.07.2014; Nature Publishing Group
• Subjects: 13/95; 631/1647/2067; 631/1647/277; 631/45/475; 631/532/2182; 82/1; 82/62; 96/10; 96/100; Animals; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical Engineering/Biotechnology; Blotting, Western - methods; Cell Differentiation; Cellular biology; Cellular proteins; Cellular signal transduction; Electrophoresis; Fluorescence; Gene expression; Green Fluorescent Proteins - biosynthesis; Innovations; Life Sciences; Monitoring methods; Neural Stem Cells - physiology; Physiological aspects; Properties; Proteins; Proteomics; Rats; Scientific imaging; Single-Cell Analysis - methods; Stem cells; Western immunoblotting; United Kingdom
• ISSN: 1548-7091; 1471-0072; 1548-7105
• DOI: 10.1038/nmeth.2992

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.