Doug Auld, Ph.D. Novartis Institutes for BioMedical Research

Find out how to analyze thousands of Western blots with single-cell resolution.

Western blots remain the method of choice to confirm protein levels in cell extracts and methods to improve the throughput and analysis of Western blot data are of high interest. This paper describes a microfluidic polyacrylamide gel (PA) procedure enabling the analysis of thousands of Western blots with single-cell resolution (see figure). The PA contains 6720 microwells, which can capture up to 4 cells per well. Following lysis, the cells are passed through the microwell by electrophoresis and protein bands can be separated over a short distance. Analysis of MW markers showed the expected log-linear relationship between MW and migration distance. Following electrophoresis the proteins were fixed using photoimmobilization to a benzophenone methacrylamide co-monomer that was cross-linked into the PA gel. Then different antibodies could be used within different blocks. In total the PA gel contained 16 microwell blocks yielding detection of up to 48 proteins (e.g., three antibodies within each block). The dynamic range was between one and two orders of magnitude with a limit of detection of ∼45 zmol (27,000 molecules). The authors applied this method to examine heterogeneity within neural stem cell (NSC) signaling that identified an isoform of nestin that may more accurately predict exit of NSCs from an immature state. The system could be used to identify such biomarkers, which could be employed in functional and phenotypic screens. 

Single-cell Western blotting. (a) The scWestern array consists of thousands of microwells (20 μm in diameter, 30 μm deep) patterned in a 30-μm-thick photoactive polyacrylamide gel seated on a glass microscope slide. The array is comprised of 16 blocks of 14 × 30 microwells (6720 in total) cast against an SU-8 photoresist master fabricated by soft lithography. E, electric field. Scale bar, 10 mm. (b) Left, wide-field micrograph of a microwell block containing 15-μm fluorescent microspheres. Scale bar, 2 mm. Right, confocal micrograph of a live EGFP-expressing NSC settled in a rhodamine-tagged gel (GEL). Scale bar, 10 μm. (c) Open-gel scWestern analysis is a 4-h, six-stage assay comprising cell settling, chemical lysis with a denaturing RIPA buffer, PAGE, UV-initiated protein immobilization onto the gel (hν, photon energy), diffusion-driven antibody probing (i.e., primary and fluorescently labeled secondary antibody probes: 1° Ab and 2° Ab*) and fluorescence imaging. (d) PAGE resolves five fluorescently labeled proteins in a 550-μm separation distance (DRO, Dronpa, 27 kDa; OVA, ovalbumin, 45 kDa; BSA, bovine serum albumin, 66 kDa; OVA′, OVA dimer, 90 kDa; BSA′, BSA dimer, 132 kDa). (e) scWestern analysis of EGFP and βTUB from a single NSC. RFU, relative fluorescence units. Distinct fluorescent dyes on each 2° Ab* enable multiplexed target analysis (EGFP, Alexa Fluor 488–labeled 2° Ab*; βTUB, Alexa Fluor 555–labeled 2° Ab*). Chemical stripping and reprobing allows multiplexed scWestern analysis.

*Abstract from Nat Methods 2014, Vol. 11: 749–760

To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ∼103 concurrent single-cell western blots (scWesterns) in ∼4 h. A microscope slide supporting a 30-mm-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.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Analytical Chemistry titled "Nanodiscs and electrospray ionization mass spectrometry: a tool for screening glycolipids against proteins." Authors of the paper are Leney AC, Fan X, Kitova EN, and Klassen JS.

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