Provides a sensitive cell-based assay of drug responsiveness to identify inhibitors relevant to neurodegenerative disease in an authentic cellular context
Reliable and simple assay doesn’t require non-physiological protein mutations or genetically engineered cell lines
Validated under a wide range of conditions and with small molecule modulators demonstrating suitability for screening compounds of potential therapeutic value
Fixed-cell assay is optimized for antibody co-localization studies
Easily quantifies aggresome and related inclusion bodies by flow cytometry
Useful for the study of neurodegenerative diseases, liver disease, toxicology studies and much more
Aggresomes are inclusion bodies of aggregated, misfolded proteins that form when the ubiquitin-proteasome protein-degradation machinery is overwhelmed. Typically, aggresomes form in response to cellular stress and provide a cytoprotective mechanism by isolating misfolded proteins, leading ultimately to their clearance by autophagy. Protein aggregate formation is furthermore a hallmark of a variety of human diseases, such as Alzheimer's, Parkinson's, Amyotrophic lateral sclerosis or alcoholic liver disease.
The PROTEOSTAT® Aggresome Detection Reagent contained in this kit is a molecular rotor dye. While in solution, free intramolecular rotation along a single central bond prevents fluorescence. The PROTEOSTAT® dye specifically intercalates into the cross-beta spine of quaternary protein structures typically found in misfolded and aggregated proteins, which will inhibit the dye’s rotation and lead to a strong fluorescence.
PROTEOSTAT® Aggresome Detection kit provides a rapid, specific and quantitative approach to label aggresomes in cells, eliminating the need of artificial protein mutations. PROTEOSTAT® dye has been validated under a wide range of conditions and with small molecule modulators, demonstrating its suitability for compound screening. Furthermore, it is suitable for multiplex co-immunofluorescence to study your target of interest in the context of autophagy and aggresome formation.
K562 cells were treated with reagents to induce ER stress and incomplete autophagy using Thapsigargin (Tg, 0.1 µM), 25, 50 and 75 µM Chloroquine (CQ) for 24 h respectively. A positive control was employed using the proteasome inhibitor, MG-132 (5 µM) for 24 h. Pelleted cells were fixed and permeabilized. Cells were then labelled for 30 min at RT with 300 µl PROTEOSTAT® Aggresome Detection reagent (Enzo Life Sciences) according to the manufacturer’s instructions and 1 µg/ml DAPI (for cell cycle determination). Relative increase in Aggresome Propensity Factor (APF) of S phase above that of G1 and that of G2m above S phase was determined for each treatment and compared to control values. ER stress inducer, Tg and 50 µM CQ was shown to both up-regulate Aggresomes more in S phase than G1 compared to controls. While proteasome inhibitor, MG-132 and 50 µM CQ was shown to down-regulate Aggresomes more in G2m than S phase than control levels.Courtesy of the Flow Cytometry Core Facility, Blizard Institute, Queen Mary University of London, London, UK.
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