From: Why and how protein aggregation has to be studied in vivo
Approach | Measurement methods | Application examples | |
---|---|---|---|
❖ Genetically encoded fusion tags | |||
✓ Fusion of the target polypeptide with a fluorescent protein or an enzyme | Reduction of fluorescence or of enzymatic activity after aggregation; detection of functional polypeptides within active IBs | Bulk cell fluorescence; fluorescence microscopy; flow cytometry; enzymatic activity | Monitoring of protein aggregation within intact cells [31]; localization of functional polypeptides within IBs [22]; formation of active IBs [16, 23, 24]; screening of aggregation inhibitors [26] |
✓ Fusion of the target polypeptide with the tetra-Cys tag | Formation of hyperfluorescent aggregates in presence of FlAsH | ||
❖ Conformational sensitive dyes | |||
✓ Thioflavin-S | Th-S fluorescence reports on amyloid-like structure of the protein aggregates | Bulk cell fluorescence; fluorescence microscopy; flow cytometry | Detection of amyloid-like aggregates within intact cells [32] |
❖ Direct spectroscopic detection of structural properties | |||
✓ FTIR | Monitoring of intermolecular β-sheet structures in IBs | Label-free intact cell (micro)spectroscopy | Monitoring of protein aggregation whithin intact cells [35, 39] |
✓ NMR | Detailed structural information of the protein embedded within IBs | Solid-state NMR of whole cells | Detection of native-like structures [43] |
❖ Aggregation sensitive reporters | |||
✓ Reporter protein under an aggregation sensitive promoter | Protein aggregation induces the expression of the reporter protein. The measured fluorescence or enzymatic activity of the reporter protein is related to the level of aggregation within cells | Enzymatic activity; fluorescence | Monitoring of protein aggregation within intact cells [37, 45] |