Measurement of aggregates

Flow cytometric-based measurement of intracellular protein aggregates

Accumulation of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. We have developed a flow cytometry-based method for quantitative measurement of fluorescently-labeled protein aggregates in detergent soluble cell extracts. The assay is used to quantify the number of fluorescent aggregates and to monitor changes in the cellular content and properties of aggregates, produced under various conditions. In addition, the assay also enables identification of aggregate components other than the primary aggregation substrate such as chaperones. The potential of this method may be extended by fluorescence-activated sorting which will facilitate the isolation of distinct protein aggregates, including those harboring proteins associated with conformation disorders. 

Determination of relative aggregate content in cells. This figure shows the fluorescence distribution of aggregates in cells expressing no reporter, Hsp104-GFP, Hsp42-mCherry, or both fluorescent reporters. The proportion of cells expressing the fluorescent reporters was determined by flow cytometry analysis (Shiber et al., 2014).