This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.
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Wang, J., Fan, H.C., Behr, B. & Quake, S.R. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 150, 402–412 (2012).
Chang, H.H., Hemberg, M., Barahona, M., Ingber, D.E. & Huang, S. Transcriptome-wide noise controls lineage choice in mammalian progenitor cells. Nature 453, 544–547 (2008).
Cohen, A.A. et al. Dynamic proteomics of individual cancer cells in response to a drug. Science 322, 1511–1516 (2008).
Pushkarsky, I. et al. Automated single-cell motility analysis on a chip using lensfree microscopy. Sci. Rep. 4, 4717 (2014).
Toriello, N.M. et al. Integrated microfluidic bioprocessor for single-cell gene expression analysis. Proc. Natl. Acad. Sci. USA 105, 20173–20178 (2008).
Duncombe, T.A., Tentori, A.M. & Herr, A.E. Microfluidics: reframing biological enquiry. Nat. Rev. Mol. Cell Biol. 16, 554–567 (2015).
Marcus, J.S., Anderson, W.F. & Quake, S.R. Microfluidic single-cell mRNA isolation and analysis. Anal. Chem. 78, 3084–3089 (2006).
Bose, S. et al. Scalable microfluidics for single cell RNA printing and sequencing. Genome Biol. 16, 120 (2015).
Fan, H.C., Fu, G.K. & Fodor, S.P.A. Combinatorial labeling of single cells for gene expression cytometry. Science 347, 1258367 (2015).
White, A.K. et al. High-throughput microfluidic single-cell RT-qPCR. Proc. Natl. Acad. Sci. USA 108, 13999–14004 (2011).
Streets, A.M. et al. Microfluidic single-cell whole-transcriptome sequencing. Proc. Natl. Acad. Sci. USA 111, 7048–7053 (2014).
Yu, M. et al. RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature 487, 510–513 (2012).
Karabacak, N.M. et al. Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat. Protoc. 9, 694–710 (2014).
Vogel, C. et al. Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line. Mol. Syst. Biol. 6, 1–9 (2010).
Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).
Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D.A. & Nolan, G.P. Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 (2005).
Rowat, A.C., Bird, J.C., Agresti, J.J., Rando, O.J. & Weitz, D.A. Tracking lineages of single cells in lines using a microfluidic device. Proc. Natl. Acad. Sci. USA 106, 18149–18154 (2009).
Hughes, A.J. et al. Single-cell western blotting. Nat. Methods 11, 749–755 (2014).
Kang, C.-C., Lin, J.-M.G., Xu, Z., Kumar, S. & Herr, A.E. Single-cell western blotting after whole-cell imaging to assess cancer chemotherapeutic response. Anal. Chem. 86, 10429–10436 (2014).
Begley, C.G. & Ellis, L.M. Drug development: raise standards for preclinical cancer research. Nature 483, 531–533 (2012).
Stadler, C. et al. Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nat. Methods 10, 315–323 (2013).
Egelhofer, T.A. et al. An assessment of histone-modification antibody quality. Nat. Struct. Mol. Biol. 18, 91–93 (2011).
Delom, F. & Chevet, E. Phosphoprotein analysis: from proteins to proteomes. Proteome Sci. 4, 15 (2006).
Hughes, A.J. & Herr, A.E. Microfluidic western blotting. Proc. Natl. Acad. Sci. USA 109, 21450–21455 (2012).
Hughes, A.J., Lin, R.K.C., Peehl, D.M. & Herr, A.E. Microfluidic integration for automated targeted proteomic assays. Proc. Natl. Acad. Sci. USA 109, 5972–5977 (2012).
Duncombe, T.A. & Herr, A.E. Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis. Lab Chip 13, 2115–2123 (2013).
Duncombe, T.A. et al. Hydrogel pore-size modulation for enhanced single-cell western blotting. Adv. Mater. 28, 327–334 (2015).
Vlassakis, J. & Herr, A.E. Effect of polymer hydration state on in-gel immunoassays. Anal. Chem. 87, 11030–11038 (2015).
Dormán, G. & Prestwich, G.D. Benzophenone photophores in biochemistry. Biochemistry 33, 5661–5673 (1994).
Burry, R.W. Controls for immunocytochemistry: an update. J. Histochem. Cytochem. 59, 6–12 (2011).
Ng, A.H.C., Chamberlain, M.D., Situ, H., Lee, V. & Wheeler, A.R. Digital microfluidic immunocytochemistry in single cells. Nat. Commun. 6, 7513 (2015).
Perfetto, S.P., Chattopadhyay, P.K. & Roederer, M. Seventeen-colour flow cytometry: unravelling the immune system. Nat. Rev. Immunol. 4, 648–655 (2004).
Shapiro, H.M. Practical Flow Cytometry (John Wiley & Sons, 2005).
Bandura, D.R. et al. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal. Chem. 81, 6813–6822 (2009).
Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).
Shi, Q. et al. Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells. Proc. Natl. Acad. Sci. USA 109, 419–424 (2012).
Lu, Y. et al. High-throughput secretomic analysis of single cells to assess functional cellular heterogeneity. Anal. Chem. 85, 2548–2556 (2013).
Stack, E.C., Wang, C., Roman, K.A. & Hoyt, C.C. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70, 46–58 (2014).
Gerdes, M.J. et al. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc. Natl. Acad. Sci. USA 110, 11982–11987 (2013).
Lu, Y. et al. Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands. Proc. Natl. Acad. Sci. 112, E607–E615 (2015).
Darmanis, S. et al. Simultaneous multiplexed measurement of RNA and proteins in single cells. Cell Rep. 14, 380–389 (2016).
Wang, D. & Bodovitz, S. Single cell analysis: the new frontier in 'omics'. Trends Biotechnol. 28, 281–290 (2010).
Zhang, W., Li, F. & Nie, L. Integrating multiple 'omics' analysis for microbial biology: application and methodologies. Microbiology 156, 287–301 (2010).
Weibrecht, I. et al. In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay. Nat. Protoc. 8, 355–372 (2013).
Brahic, M., Haase, A.T. & Cash, E. Simultaneous in situ detection of viral RNA and antigens. Proc. Natl. Acad. Sci. USA 81, 5445–5448 (1984).
Fienberg, H.G., Simonds, E.F., Fantl, W.J., Nolan, G.P. & Bodenmiller, B. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry 81A, 467–475 (2012).
Behbehani, G.K., Bendall, S.C., Clutter, M.R., Fantl, W.J. & Nolan, G.P. Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry 81A, 552–566 (2012).
Zhang, Y. et al. Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells. Anal. Chem. 87, 9761–9768 (2015).
Xue, M. et al. Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells. J. Am. Chem. Soc. 137, 4066–4069 (2015).
Rodbard, D., Kapadiia, G. & Chrambach, A. Pore gradient electrophoresis. Anal. Biochem. 157, 135–157 (1971).
Li, J.J., Bickel, P.J. & Biggin, M.D. System wide analyses have underestimated protein abundances and the importance of transcription in mammals. PeerJ 2, e270 (2014).
Tong, J. & Anderson, J.L. Partitioning and diffusion of proteins and linear polymers in polyacrylamide gels. Biophys. J. 70, 1505–1513 (1996).
Morris, C.J. & Morris, P. Molecular-sieve chromatography and electrophoresis in polyacrylamide gels. Biochem. J. 124, 517–528 (1971).
Tanaka, T. et al. Mechanical instability of gels at the phase transition. Nature 325, 796–798 (1987).
Go, Y.M. & Jones, D.P. Redox compartmentalization in eukaryotic cells. Biochim. Biophys. Acta 1780, 1273–1290 (2008).
Margolis, J. & Kenrick, K.G. Polyacrylamide gel-electrophoresis across a molecular sieve gradient. Nature 214, 1334–1336 (1967).
Ebersole, R.C. & Foss, R.P., inventors. Porosity gradient electrophoresis gel. U.S Pat. 4,704,198 (1987).
Swinney, K. & Bornhop, D.J. Quantification and evaluation of Joule heating in on-chip capillary electrophoresis. Electrophoresis 23, 613–620 (2002).
Petersen, N.J., Nikolajsen, R.P.H., Mogensen, K.B. & Kutter, J.P. Effect of Joule heating on efficiency and performance for microchip-based and capillary-based electrophoretic separation systems: A closer look. Electrophoresis 25, 253–269 (2004).
Pan, Y., Duncombe, T.A., Kellenberger, C.A., Hammond, M.C. & Herr, A.E. High-throughput electrophoretic mobility shift assays for quantitative analysis of molecular binding reactions. Anal. Chem. 86, 10357–10364 (2014).
Torsvik, A. et al. U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells. Cancer Med. 3, 812–824 (2014).
Timerman, D. & Yeung, C.M. Identity confusion of glioma cell lines. Gene 536, 221–222 (2014).
Madren, S.M. et al. Microfluidic device for automated synchronization of bacterial cells. Anal. Chem. 84, 8571–8578 (2012).
Na, S. et al. Rapid signal transduction in living cells is a unique feature of mechanotransduction. Proc. Natl. Acad. Sci. USA 105, 6626–6631 (2008).
Henjes, F. et al. Strong EGFR signaling in cell line models of ERBB2-amplified breast cancer attenuates response towards ERBB2-targeting drugs. Oncogenesis 1, e16 (2012).
Giddings, J.C. Unified Separation Science 97–101 (John Wiley & Sons, 1991).
Bendall, S.C., Nolan, G.P., Roederer, M. & Chattopadhyay, P.K. A deep profiler's guide to cytometry. Trends Immunol. 33, 323–332 (2012).
Ornatsky, O.I. et al. Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Anal. Chem. 80, 2539–2547 (2008).
Albayrak, C. et al. Digital quantification of proteins and mRNA in single mammalian cells. Mol. Cell 61, 914–924 (2016).
Leuchowius, K.-J., Weibrecht, I. & Söderberg, O. In situ proximity ligation assay for microscopy and flow cytometry. Curr. Protoc. Cytom. Chapter 9 Unit 9.36 (2011).
Gawad, S., Schild, L. & Renaud, P.H. Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing. Lab Chip 1, 76–82 (2001).
Porichis, F. et al. High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat. Commun. 5, 5641 (2014).
Research reported in this publication was supported in part by the National Cancer Institute of the National Institutes of Health (R21CA183679 to A.E.H.), a New Innovator Award from the National Institutes of Health (1DP2OD007294 to A.E.H.), an NSF CAREER award from the National Science Foundation (CBET-1056035 to A.E.H.), a Diversity Supplement from the National Institutes of Health (to E.S.) and National Science Foundation Graduate Research Fellowships (DGE 1106400 to K.A.Y., J.V. and T.A.D.). We are grateful to S. Kumar's laboratory in the Department of Bioengineering, University of California, Berkeley, for providing the U373 MG and U373-GFP cell lines. We acknowledge the helpful feedback from students enrolled in the 2015 Single Cell Analysis course at Cold Spring Harbor Laboratory.
All co-authors are co-inventors on intellectual property related to the device and assay described here and may benefit from royalties from licensing.
Integrated supplementary information
Example wafer mold design for the scWB. The wafers are fabricated using standard photolithography techniques with SU-8 2025 photoresist. As SU-8 2025 is a negative photoresist, regions of the mask containing features to be polymerized (e.g. the micropillars) should be transparent. The photomasks are printed on mylar.
The scWB electrophoresis chamber is fabricated out of Acrylonitrile butadiene styrene with a fused deposition molding 3D printer (e.g. MakerBot Replicator 2x). The chamber was printed with the cavity facing up and with a raft composed of dissolvable filament. The solid models was produced in SolidWorks and preprocessed in MakerWare.
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Kang, C., Yamauchi, K., Vlassakis, J. et al. Single cell–resolution western blotting. Nat Protoc 11, 1508–1530 (2016) doi:10.1038/nprot.2016.089
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