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Table 1 Methods for the study of protein aggregation in intact cells

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]