Detection of Isoforms Differing by a Single Charge Unit in Individual Cells

To measure protein isoforms in individual mammalian cells, we report single‐cell resolution isoelectric focusing (scIEF) and high‐selectivity immunoprobing. Microfluidic design and photoactivatable materials establish the tunable pH gradients required by IEF and precisely control the transport and handling of each 17‐pL cell lysate during analysis. The scIEF assay resolves protein isoforms with resolution down to a single‐charge unit, including both endogenous cytoplasmic and nuclear proteins from individual mammalian cells. Electrophoretic cytometry: A multilayer, patterned hydrogel device supports isoelectric focusing to separate protein isoforms from single cells (scIEF). All preparative and analytical steps, including cell isolation, lysis, protein separation, UV‐actuated blotting, and immunoprobing, are performed on the device. Protein isoforms with single‐charge differences are resolved, blotted, and detected by immunoprobing.