In Situ Single-Cell Western Blot on Adherent Cell Culture
| Autor: | Amy E. Herr, Isao Naguro, Yizhe Zhang |
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| Rok vydání: | 2019 |
| Předmět: |
Lysis
Cell Blotting Western Cell Culture Techniques 010402 general chemistry 01 natural sciences Catalysis Article HeLa Western blot medicine Humans Protein phosphorylation Polyacrylamide gel electrophoresis medicine.diagnostic_test biology 010405 organic chemistry Chemistry General Chemistry General Medicine Trypsin biology.organism_classification Molecular biology 0104 chemical sciences Blot medicine.anatomical_structure medicine.drug |
| Zdroj: | Angew Chem Int Ed Engl |
| ISSN: | 1521-3773 |
| Popis: | Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses. |
| Databáze: | OpenAIRE |
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