Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

Single-cell western blotting differentiates truncated isoforms/CTFs from full-length HER2 protein in primary HER2-positive breast tumor biopsies. a The microfluidic scWB device is designed to assay the expression of different HER2 forms in heterogeneous tumor cells derived from dissociated HER2-positive breast cancer tumors. The scWB workflow includes five steps: gravity-based settling of single cells into each of an array of microwells, in situ chemical cell lysis, polyacrylamide gel electrophoresis (PAGE) of each single-cell lysate, UV-activated protein immobilization, and finally in-gel immunoprobing. With PAGE, the scWB can resolve truncated (t-erbB2s) from full-length HER2 oncoproteins (p185HER2) in each 1-mm long separation distance. PAGE physically separates background signal from the target-protein peaks. b False-color fluorescence micrographs and corresponding intensity plots resolve smaller t-erbB2s (~95？kDa) from larger p185HER2 oncoproteins (~185？kDa) in positive controls cells (genetically modified CHO cells; CHO/p185 and CHO/t-erbB2; 15-sec PAGE in 7%T, 2.6%C gel). Immunoprobing of actinin is an internal electromigration control. AFU Arbitrary Fluorescence Unit. c Dot plot shows a significant difference in the mean electromigration distance for p185HER2 versus t-erbB2s (two-tailed t-test, preplicate 1？<？1？×？10？10, labeled as *; preplicate 2？<？1？×？10？10, labeled as **). All measurements outside 4 times the standard deviation from each mean were discarded. d, False-color fluorescence micrographs and intensity plots show that HER2-2G11 antibody, which recognizes the ECD of HER2, detects the p185HER2 protein peak, but not the t-erbB2 protein peak detected using HER2-3B5 immunoprobing of the same BT474 cells. e and f Scatter plots and intensity plots of relative p185HER2 and t-erbB2 expression in a population of BT474 cell