AutoBlot: deterministic single-cell western blotting reveals proteomic diversity in rare cell populations

Single-cell western blotting (scWB) provides quantitative, molecular-mass-resolved protein measurements but relies on Poisson-limited gravity settling, requiring ∼10 ⁶ starting cells with <37% microwell occupancy. AutoBlot replaces stochastic loading with piezoelectric dispensing, achieving ∼98% occupancy and deterministic cell-bead co-isolation from as few as 10,000 starting cells. Profiling patient-derived breast organoids (PDOs), we detect BRCA1-associated proteomic shifts, menopausal-status-dependent ER expression differences, and systematic differences between surface and intracellular lineage classification of mammary epithelial cells in organoid culture.