Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Removal of misfolded proteins in the cell is majorly regulated by the cellular ubiquitin-proteasome system, wherein the protein is first recognized and tagged by ubiquitin, a small regulatory protein, and trafficked to the proteasome, a multiprotein complex, for hydrolysis and clearance.

Impaired ubiquitin-proteosome system activity, a common occurrence in neurodegenerative diseases, can cause misfolded proteins to accumulate within the cells, forming insoluble protein aggregates with cytotoxic effects.

To visualize and quantify protein aggregation in vitro, obtain a suspension of transduced mammalian cells - expressing a fluorescent-labeled mutant protein that adopts a misfolded conformation with exposed hydrophobic residues - in suitable media.

Pipette increasing concentrations of a proteasome inhibitor to the wells of a black multi-well plate to minimize background fluorescence during imaging. Seed the mammalian cell suspension into the wells. Incubate to allow the cells to adhere to the plate bottom and proliferate.

Once inside, the proteasome inhibitor binds to the proteolytic sites of the proteasome and blocks its activity, causing the expressed mutant proteins to accumulate, which then interact via their exposed hydrophobic residues and form cytoplasmic aggregates, eventually leading to cell death.

Add a fluorescent DNA-binding dye to stain the cell nuclei. Image the plate. The fluorescent mutant protein aggregates appear dispersed within the unstained cytoplasm.

Wells containing higher proteasome inhibitor concentrations exhibit higher protein aggregate numbers to cell count ratio than wells with lower proteasome inhibitor concentrations.