title: Single-cell western blotting.
abstract: To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼10(3) concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (∼200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.
authors.affiliation: 1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] [4].
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Hughes
authors.affiliation: 1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA. [4].
authors.forename: Dawn P
authors.initials: DP
authors.lastname: Spelke
authors.affiliation: 1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.
authors.forename: Zhuchen
authors.initials: Z
authors.lastname: Xu
authors.affiliation: 1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.
authors.forename: Chi-Chih
authors.initials: CC
authors.lastname: Kang
authors.affiliation: 1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA. [4] Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA. [5] Helen Wills Neuroscience Institute, UC Berkeley, Berkeley, California, USA.
authors.forename: David V
authors.initials: DV
authors.lastname: Schaffer
authors.affiliation: 1] Department of Bioengineering, University of California (UC) Berkeley, Berkeley, California, USA. [2] California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. [3] The UC Berkeley-UC San Francisco Graduate Program in Bioengineering, UC Berkeley, Berkeley, California, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002454:Cell Differentiation; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D058953:Neural Stem Cells / Q000502:physiology; D051381:Rats; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins