Subcellular western blotting of single cells

作     者：Kevin A.Yamauchi Amy E.Herr 

作者机构：Department of BioengineeringUniversity of CaliforniaBerkeleyCA 94720USA The UC Berkeley—UCSF Graduate Program in BioengineeringUniversity of CaliforniaBerkeleyCA 94720USA 

出 版 物：《Microsystems & Nanoengineering》 (微系统与纳米工程（英文）)

年 卷 期：2017年第3卷第1期

页      面：405-413页

核心收录：

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

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) 

主　　题：cytometry microfluidic design proteomics single-cell analysis 

摘      要：Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells,antibody probe cross-reactivity and fixation artifacts remain confounding *** 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 *** confer precision fluidic control,we describe a passive multilayer microdevice that leverages the rapid transport times afforded by *** 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 ***,we lyse the intact nucleus and western blot the nuclear *** 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 ***-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in *** 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 *** chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells,thus providing insight into protein signaling in heterogeneous cell populations.