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    188宝金博页面版: Single-cell western blotting(单细胞免疫印迹)

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    内容提示： Single-cell western blottingAlex J. Hughes 1,2,6,7 , Dawn P. Spelke 1,2,3,7 , Zhuchen Xu 1,2 , Chi-Chih Kang 1,2 , David V. Schaffer 1,2,3,4,5 , and Amy E. Herr 1,2,31 Department of Bioengineering, University of California, Berkeley (UC Berkeley), California, USA2 California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA3 The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA4 Department of Chemical Engineering, UC Berkele...

    文档格式：PDF | 页数：27 | 浏览次数：86 | 上传日期：2020-10-11 11:12:58 | 文档星级：
    Single-cell western blottingAlex J. Hughes 1,2,6,7 , Dawn P. Spelke 1,2,3,7 , Zhuchen Xu 1,2 , Chi-Chih Kang 1,2 , David V. Schaffer 1,2,3,4,5 , and Amy E. Herr 1,2,31 Department of Bioengineering, University of California, Berkeley (UC Berkeley), California, USA2 California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA3 The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA4 Department of Chemical Engineering, UC Berkeley, Berkeley, California, USA5 Helen Wills Neuroscience Institute, UC Berkeley, Berkeley, California, USAAbstractTo measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~10 3 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsCorrespondence should be addressed to D.V.S (schaffer@berkeley.edu) or A.E.H. (aeh@berkeley.edu).6 Present address: Department of Pharmaceutical Chemistry, UC San Francisco, San Francisco, California, USA.7 These authors contributed equally to this work.AUTHOR CONTRIBUTIONSA.J.H. and A.E.H. designed the scWestern. A.J.H., A.E.H., D.P.S., and D.V.S. designed experiments. A.J.H., Z.X., and C.-C. K. performed scWesterns and calibration experiments. D.P.S. performed NSC culture, stimulation and differentiation; conventional western blots, flow cytometry, and plate-based immunocytochemistry. A.J.H. and D.P.S performed in-microwell immunocytochemistry. A.J.H., D.P.S., and Z.X. performed confocal microscopy. Z.X. and A.J.H. designed software and performed analysis for cell/microwell scoring, immunoprobing quality control, and fluorescence quantitation. Z.X. performed fluid dynamics simulations. Validation data were analyzed by A.J.H, D.P.S., and Z.X. All authors wrote the manuscript.COMPETING INTERESTS STATEMENTA.J.H., Z.X., C.-C.K., and A.E.H. are inventors on pending patents related to scWestern blot methods. A.E.H. holds equity interest in Zephyrus Biosciences.HHS Public AccessAuthor manuscriptNat Methods. Author manuscript; available in PMC 2015 January 01.Published in final edited form as:Nat Methods. 2014 July ; 11(7): 749–755. doi:10.1038/nmeth.2992.Author Manuscript Author Manuscript Author Manuscript Author Manuscript

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