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.To enhance selecti...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.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.To confer precision fluidic control,we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization.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.Subsequently,we lyse the intact nucleus and western blot the nuclear lysate.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.Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry.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).Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence.This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells,thus providing insight into protein signaling in heterogeneous cell populations.展开更多
基金KAY is a National Science Foundation Graduate Research Fellow(DGE 1106400)This work was supported by the National Science Foundation CAREER award(CBET-1056035 to AEH)National Institutes of Health(R01CA203018 to AEH).
文摘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.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.To confer precision fluidic control,we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization.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.Subsequently,we lyse the intact nucleus and western blot the nuclear lysate.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.Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry.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).Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence.This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells,thus providing insight into protein signaling in heterogeneous cell populations.
