Single Cell Western Blot Service
Single cell western blot is a technique for analyzing proteins at the level of a single cell using fluorescent antibodies after Western blotting on a microchip.
Introduction to Single Cell Western Blot
Single cell western blot (scWestern) is a highly scalable multiplexed method for measuring heterogeneity in protein content between cells. Each microwell on the scWestern platform can hold a single cell. The cells are lysed, and the proteins are electrophoretically loaded into the surrounding gel. After separation, the proteins are photo-immobilized using an ultraviolet-active benzophenone methacrylamide comonomer encapsulated within the gel. After the proteins are bound to primary and secondary antibodies, they are imaged.
Fig.1 Diagram of single cell western blot workflow. (Labib, 2020)
Advantages of Single Cell Western Blot
- Suitable for small biospecimens (2-1000 cells).
- Suitable for analysis of cell surface and cytoplasmic proteins.
- Capable of analyzing protein isoforms and truncated proteins.
- Highly selective analysis owing to electrophoresis or isoelectric-focusing steps.
Published Data
| Paper Title | Single-cell western blotting |
| Journal | Nature Methods |
| Published | 2014 |
| Abstract | To measure cell-to-cell variation in protein-mediated functions, we devised a method for performing 103 simultaneous single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30-µm-thick photoactive polyacrylamide gel enables western blotting, including the settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody detection. We utilized this scWestern technique to track the single-cell differentiation and mitogen-induced responses of rat neural stem cells. scWestern quantified target proteins despite off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds <30,000 molecules, and supported analyses of low starting cell numbers (~200) when combined with FACS. The scWestern overcomes the antibody fidelity and sensitivity limitations of other single-cell protein analysis methods and is a versatile instrument for the study of complex cell populations at single-cell resolution. |
| Result |
As a second design principle, they optimize for short-separation-distance PA-gel electrophoresis to achieve a dense scWestern array (PAGE). In order to initiate electrophoresis following cell lysis, an electric field is applied across a submerged scWestern slide, electrophoresizing proteins through the microwell walls and into the thin PA-gel sheet. To characterize this process, a ladder of fluorescently labeled purified proteins was evaluated, which segregate into microwells (Fig. 2A). Then, they analyze EGFP and TUB from a single neural stem cell using scWestern (Fig. 2B).
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Fig.2 Single cell western blotting. (Hughes, 2014)
Single Cell Western Blot Service
At Creative Biolabs, we can profile the target protein expression heterogeneity and cell type heterogeneity for your samples with our single cell western blot services which would go undetected by traditional western blot. For more information, please contact us.
References
- Labib, M.; Kelly, S.O. Single-cell analysis targeting the proteome. Nature Review Chemistry. 2020, 4: 143-158.
- Hughes, A.J.; et al. Single-cell western blotting. Nature Methods. 2014, 11: 749-755.
