Heterogeneity is inherent to cellular processes, including differentiation. Western blotting is a sensitive method for detecting and quantifying specific proteins within a mixed sample. Here we report on a single cell western blotting method capable of measuring, with high specificity, cell-to-cell protein heterogeneity within complex populations of cells. This method permits simultaneous assays of approximately 2000 individual cells in less than four hours. We applied the method to study immature neural stem cells and their differentiation over a six day period under conditions that yielded both astroctyes and neurons. The method successfully reported the presence of proteins associated with neural differentiation and identified a variant of the protein nestin that was sharply downregulated with development.

This single cell Western blotting method 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.