Abstract
Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.
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References
Chang HH, Hemberg M, Barahona M et al (2008) Transcriptome-wide noise controls lineage choice in mammalian progenitor cells. Nature 453(7194):544–547
Dalerba P, Kalisky T, Sahoo D et al (2011) Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nat Biotechnol 29(12):1120–1127
Balic M, Williams A, Lin H et al (2013) Circulating tumor cells: from bench to bedside. Annu Rev Med 64:31–44
Bendall SC, Simonds EF, Qiu P et al (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332(6030):687–696
Tay S, Hughey JJ, Lee TK et al (2010) Single-cell NF-[kgr] B dynamics reveal digital activation and analogue information processing. Nature 466(7303):267–271
Altschuler SJ, Wu LF (2010) Cellular heterogeneity: do differences make a difference? Cell 141(4):559–563
Ciaccio MF, Wagner JP, Chuu C et al (2010) Systems analysis of EGF receptor signaling dynamics with microwestern arrays. Nat Methods 7(2):148–155
Hughes AJ, Herr AE (2012) Microfluidic western blotting. Proc Natl Acad Sci 109(52):21450–21455
Ashton RS, Conway A, Pangarkar C et al (2012) Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling. Nat Neurosci 15(10):1399–1406
Sevecka M, MacBeath G (2006) State-based discovery: a multidimensional screen for small-molecule modulators of EGF signaling. Nat Methods 3(10):825–831
Hughes AJ, Spelke DP, Xu Z et al (2014) Single-cell western blotting. Nat Methods 11(7):749–755
Wood DK, Weingeist DM, Bhatia SN et al (2010) Single cell trapping and DNA damage analysis using microwell arrays. Proc Natl Acad Sci 107(22):10008–10013
Cao X, Shoichet MS (2002) Photoimmobilization of biomolecules within a 3-dimensional hydrogel matrix. J Biomater Sci Polym Ed 13(6):623–636
Hughes AJ, Herr AE (2010) Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography. Anal Chem 82(9):3803–3811
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Sinkala, E., Herr, A.E. (2015). Single-Cell Western Blotting. In: Singh, A., Chandrasekaran, A. (eds) Single Cell Protein Analysis. Methods in Molecular Biology, vol 1346. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2987-0_1
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DOI: https://doi.org/10.1007/978-1-4939-2987-0_1
Publisher Name: Humana Press, New York, NY
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