The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors.
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