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Single-cell western blotting
Alex J Hughes1,2,6,7, Dawn P Spelke13,7, Zhuchen Xu1,2, Chi-Chih Kang1,2, David V Schaffer15 & Amy E Herr13
npg 201 4 Nature America, Inc. All rights reserved.
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-mm-thick photoactive polyacrylamidegel 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 quantied target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholdsof <30,000 molecules, and supported analyses of low starting cell numbers (~200) when integrated with FACS. The scWestern overcomes limitations of antibody delity and sensitivity in other single-cell protein analysis methods and constitutesa versatile tool for the study of complex cell populations at single-cell resolution.
Heterogeneity is inherent in cellular processes including stem cell differentiation1,2, development3, cancer4,5, pharmaceutical efficacy6 and immune response7. Owing in large part to recent technological advances, genomic and transcriptomic studies of cell-to-cell heterogeneity are flourishing4,8. However, recent single- cell and population-wide studies comparing transcriptomes to proteomes in microorganismal and mammalian cells found only mild correlations between mRNA and protein expression911.
Therefore, to fully understand diverse and often rare behaviors in complex cell populations, researchers need analytical tools that are optimized for protein analysis of many cells, offer single-cell resolution, provide quantitative and highly specific detection of target proteins, and do not employ labels that may perturb protein and cell function12.
Single-cell proteome-wide studies are currently limited to readouts from synthetic fluorescent-protein fusion libraries9,11,
which, though illuminating, are challenging to generate and can potentially perturb protein function. Single-cell protein immunoassays (for example, flow cytometry7 and immunocytochemistry (ICC)13) have proved immensely important for assessing cell-to-cell heterogeneity, yet existing methods depend
on analyte discrimination with antibody probes that often have limited specificity. This dependence on antibody probe quality restricts assay performance, as cross-reactivity can create misleading background signals that are difficult to correct for1416, even with careful controls17,18. This vulnerability broadly affects antibody-based assays (such as ELISAs and protein micro-arrays14). The widely used...