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.

Figure 1

Single-cell western blotting. (a) The scWestern array consists of thousands of microwells (20 µm 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 (6,720 in total) cast against an SU-8 photoresist master fabricated by soft lithography. E: electric field. Scale bar: 10 mm. (b) Widefield micrograph of a microwell block containing 15 µm fluorescent microspheres (scale bar: 2 mm), and confocal micrograph of a live EGFP-expressing neural stem cell (NSC) settled in a rhodamine-tagged gel (GEL, scale bar: 10 µm). (c) Open-gel scWestern analysis is a 4 hour, 6 stage assay comprised of: cell settling, chemical lysis with a denaturing RIPA buffer, polyacrylamide gel electrophoresis (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 5 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 β-tubulin (βTUB) from a single NSC (RFU: relative fluorescence units). Distinct fluorescent dyes on each secondary antibody enable multiplexed target analysis (EGFP: Alexa Fluor 488-labeled secondary antibody, βTUB: Alexa Fluor 555−). Chemical stripping and re-probing allows multiplexed scWestern analysis. Antibody details for all figures are in Online Methods.

Figure 2

scWestern blotting of NSCs. (a) 420 concurrent scWesterns of EGFP-expressing NSCs for βTUB (Alexa Fluor 647-labeled secondary antibody, 2° Ab*) and EGFP (Alexa Fluor 555−). Bright-field imaging determines cells/microwell. (b) Top-right: scWestern fluorescence for 4,128 separations by cells/microwell. Bottom-left: area under the curve for βTUB with (above) running average of cells/microwell (window size = 30 microwells). Microwells indexed from left-hand to right-hand side of array. Bottom-right: Fit of fluorescence distribution for single cells to gamma distribution stemming from Poissonian mRNA production and exponentially distributed protein burst sizes f(x)=(x^a−1e^−x/b)/(Γ(a)b^a); x = total probed band fluorescence, a = μ_p²/σ_p² = 14.8, b = σ_p²/μ_p = 1.6×10⁵ AFU, μ_p = mean band fluorescence, σ_p² = variance in band fluorescence, Γ is the gamma function. (c) EGFP fluorescence for 1 and 0 cell/microwell blots compared to flow cytometry of fixed NSCs (EGFP transfected, +ve; untransfected, −ve), note arcsinh-transformed scales (Online Methods). Technical noise estimated from scWesterns with 0 cell/microwell in a sparsely cell-seeded region (separations 4,100–4,128, 0*; Supplementary Note 5). Fraction EGFP+ cells is mean ± S.D. for n = 3. (d) Signal-to-noise estimates limit of detection at 27,000 molecules using purified EGFP through direct and indirect methods (see text, mean ± S.D., n = 3 regions of interest per dot blot). Bottom, linear indirect calibration curves for purified standards (mean ± S.D., n = 3 regions of interest per dot blot) span physiologically relevant βTUB and ERK concentrations (concentration in a probed band estimated from in-cell concentrations^,).

Figure 3

scWesterns capture fibroblast growth factor-2 (FGF-2) signaling dynamics. (a) Fluorescence micrographs of scWesterns for ERK, pERK, MEK, pMEK in NSCs, with β-tubulin (βTUB) and EGFP ladder. For each target pair, EGFP image is from a distinct separation in the same microwell array row. 103 kDa off-target peak (via pERK antibody) does not coincide with ERK band. Secondary antibodies were Alexa Fluor 555-labeled (except EGFP; Alexa Fluor 488−) in order: pERK, ERK and EGFP co-probe, βTUB, pMEK, MEK; with stripping between probings. (b) Fraction off-target pERK versus total fluorescence for 1,117 scWesterns at timepoints from (d) and (e). (c) Conventional western blots (20 ng/ml FGF-2), cropped to show regions of interest. Full-length blots are presented in Supplementary Fig. 20. (d) Fold-change of pERK and pMEK to total ERK and MEK, respectively, with signals below technical noise indicated. Note arcsinh-transformed scales. Overlay from conventional western blot densitometry. ***P < 0.001, Mann-Whitney. n = 186, 186, 57, 236, 278, and 208 scWesterns for 0, 5, 12, 20, 30, and 60 min time-points, respectively. (e) Fold-change from (d) with spatial density contours. (f) Immunocytochemistry co-probing for pERK-ERK and pMEK-MEK pairs; Alexa Fluor 555-labeled secondary phospho-antibodies and Alexa Fluor 647-labeled secondary total-antibodies. pERK:ERK: n = 160, 115, 186, 158, 172, and 197 cells for 0, 5, 12, 20, 30, and 60 min time-points, respectively. pMEK:MEK: n = 184, 216, 220, 223, 223, and 270 cells for 0, 5, 12, 20, 30, and 60 min time-points, respectively.

Figure 4

scWesterns track NSC lineage commitment during differentiation. (a) Immunocytochemistry micrographs of mixed NSC differentiation cultures at days 0 and 6 for stem cell (nestin, NEST; SOX2) and differentiation markers (βIII-tubulin, βIIITUB; glial fibrillary acidic protein, GFAP). Scale bar: 50 µm. (b) Micrographs of NSCs in scWestern microwells, fixed and stained as in (a). (c) Confocal images of fixed and stained stem (NEST+, SOX2+), neuron (βIIITUB+) and astrocyte (GFAP+) cells in a rhodamine-tagged gel (GEL). Scale bar: 10 µm. (d) Inverted fluorescence micrographs of scWesterns. SOX2 (Alexa Fluor 555-labeled secondary antibody) and NEST (α and β isoforms, Alexa Fluor 488−) were co-probed in separate blocks as GFAP (Alexa Fluor 555−) and βIIITUB (Alexa Fluor 488−); both block sets were stripped and co-probed for β-tubulin (βTUB, Alexa Fluor 555−) and EGFP (Alexa Fluor 488−). Image sets from each day are the same separation, except EGFP images, which are from the same microwell array row as the corresponding image set. (e) Cropped conventional western blots at differentiation days 0 and 6. Full-length blots are presented in Supplementary Fig. 29. (f) scWestern fluorescence normalized by βTUB (arbitrary units). Note arcsinh-transformed scales. Spatial density indicated by contours. scWestern blot NSC marker sample sizes, n = 189, 353, 175, and 274 for time-points at 0, 1, 2, and 6 days, respectively. Differentiation marker sample sizes, n = 178, 253, 303, 280 for time-points at 0, 4, 5, and 6 days, respectively. Data are one of two biological replicates performed.