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Merck

3D projection electrophoresis for single-cell immunoblotting.

Nature communications (2020-12-06)
Samantha M Grist, Andoni P Mourdoukoutas, Amy E Herr
ABSTRACT

Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (xy) for cell isolation and lysis. Single-cell lysate in each microwell is "electrophoretically projected" into the 3rd dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is > 2.5 cells/s (70× faster than published serial sampling), with 25 immunoblots/mm2 device area (>10× increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cells. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-PTBP1 antibody produced in mouse, clone 3H8, purified immunoglobulin, buffered aqueous solution
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Acrylamide/Bis-acrylamide, 30% solution, BioReagent, suitable for electrophoresis, 37.5:1
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Dichlorodimethylsilane, ≥99.5%
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Sucrose, for molecular biology, ≥99.5% (GC)
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Triton X-100, laboratory grade
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N,N,N′,N′-Tetramethylethylenediamine, BioReagent, suitable for electrophoresis, ≥99.0%
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