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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Product Details
| Applications: | Flow Cytometry, Fluorescence microscopy, Fluorescent detection
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| Application Notes: | The PROTEOSTAT® Aggresome detection kit assay is potentially applicable to the identification of small molecules that inhibit aggresome formation as well as immuno-localization studies between aggregated protein cargo and the various proteins implicated in aggresome formation, such as histone deacetylase 6, parkin, ataxin-3, dynein motor complex and ubiquilin-1. |
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| Quality Control: | A sample from each lot of PROTEOSTAT® Aggresome detection kit is used to stain Jurkat cells and analyzed by flow cytometry, using the procedures described in the user manual. The aggresome activity factor (AAF) values for the samples are greater than 25. |
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| Quantity: | For ENZ-51035-0025:
25 flow cytometry assays or 50 microscopy assays
For ENZ-51035-K100:
100 flow cytometry assays or 200 microscopy assays |
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| Use/Stability: | With proper storage, the kit components are stable for one year from date of receipt. |
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| Handling: | Protect from light. Avoid freeze/thaw cycles. |
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| Shipping: | Shipped on Blue Ice |
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| Short Term Storage: | -20°C |
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| Long Term Storage: | -80°C |
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| Contents: | PROTEOSTAT®Aggresome detection reagent
Hoechst 33342 Nuclear stain
Proteasome Inhibitor (MG-132)
10X Assay Buffer |
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| Technical Info/Product Notes: | The PROTEOSTAT® Aggresome Detection Kit is a member of the CELLESTIAL® product line, reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications. CELLESTIAL® reagents and kits are optimal for use in demanding imaging applications, such as confocal microscopy, flow cytometry and HCS, where consistency and reproducibility are required.
Featured In:
High-throughput image-based aggresome quantification (SLAS Discov 2020; 25:783-791).
Application: Optimized to monitor aggresome formation in a miniaturized, automated, and quantitative phenotypic assay. The approach is validated by screening a chemical library of 1280 compounds.
Application Note:
A Red-emitting Fluorescent Probe for Rapid Detection of Protein and Peptide Aggregates in Post-mortem Human Brain Tissue Sections from Patients Diagnosed with Alzheimer’s and Parkinson’s Disease
Monitoring the Accumulation and Clearance of Exogenously Introduced Beta-amyloid in a Cell-Based Model of Alzheimer’s Disease by Fluorescence Microscopy and Fluorescence Microplate Assay
Towards Understanding the Molecular Basis of Parkinson’s Disease: Cell-based Model of Mitophagy and Aggresome Accumulation
Detection of bacterial aggregation by flow cytometry
Cell-Based Screening of Focused Bioactive Compound Libraries: Assessing Small Molecule Modulators of the Canonical Wnt Signaling and Autophagy-Lysosome Pathways
Cited samples:
For an overview on cited samples please click here.
Enzo and PROTEOSTAT® are trademarks of Enzo Life Sciences, Inc. Several of Enzo’s products and product applications are covered by US and foreign patents and patents pending. |
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| Regulatory Status: | RUO - Research Use Only |
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Product Literature References
Cell-Instructive Surface Gradients of Photoresponsive Amyloid-like Fibrils: A.M. Bender, et al.; ACS Biomater. Sci. Eng. 7, 4798 (2021), Abstract;
Dual Targeting of Endoplasmic Reticulum by Redox-Deubiquitination Regulation for Cancer Therapy: B. Cai, etr al.; Int. J. Nanomedicine 16, 5193 (2021), Abstract;
Inhibition of cardiac PERK signaling promotes peripartum cardiac dysfunction: T. Shimizu, et al.; Sci. Rep. 11, 18687 (2021), Abstract;
New evidences of ubiquitin-proteasome system activity in human sperm: J.V. Silva, et al.; Biochim. Biophys. Acta Mol. Cell. Res. 1868, 118932 (2021), Application(s): Microscopy; detection of aggresome-like inclusion bodies in human sperm, Abstract;
Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol: C.T. Hellwig, et al.; Cell Death Differ. (2021), Abstract;
Sodium sulfite causes gastric mucosal cell death by inducing oxidative stress: O. Moeri, et al.; Free Radic. Res. (2021), Abstract;
4-Phenylbutyrate ameliorates apoptotic neural cell death in Down syndrome by reducing protein aggregates: K. Hirata, et al.; Sci. Rep. 10, 14047 (2020), Abstract; Full Text
Cytokinin-induced protein synthesis suppresses growth and osmotic stress tolerance: S.S. Karunadasa, et al.; New Phytol. 227, 50 (2020), Application(s): Confocal microscopy; A. thaliana seedlings, Abstract;
DMSO impairs the transcriptional program for maternal-to-embryonic transition by altering histone acetylation: M.H. Kang, et al.; Biomaterials 230, 119604 (2020), Application(s): Confocal microscopy using mouse embryos, Abstract;
Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients: K.Y. Kim, et al.; Transl. Neurodegener. 9, 23 (2020), Abstract; Full Text
Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease: C.C. Tao, et al.; Cell Death Differ. 27, 192 (2020), Application(s): Visualisation of amyloid plaques on mouse brain sections, Abstract; Full Text
hnRNPDL phase separation is regulated by alternative splicing and disease-causing mutations accelerate its aggregation: C. Batlle, et al.; Cell Rep. 30, 1117 (2020), Application(s): Fluorescence microscopy using aggregated proteins in solution, Abstract;
Intravaginal poly-(D, L-lactic-co-glycolic acid)-(polyethylene glycol) drug-delivery nanoparticles induce pro-inflammatory responses with Candida albicans infection in a mouse model: T.T. Lina, et al.; PLoS One 15, e0240789 (2020), Abstract; Full Text
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