Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor
Published: April 30, 2023
Abstract
Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.
Protocol
1. Lentivirus production NOTE: The production and manipulation of lentiviral vectors was carried out according to the National Institutes of Health (NIH) guidelines for research involving recombinant DNA. The plasmid encoding the wild-type and A4V mutant SOD1 tagged with enhanced YFP (SOD1WT-YFP and SOD1A4V-YFP) are used. Both gene fusion products were amplified using the PCR primer pair 5′-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3′ and 5′-TAGCGG CCGCTACTTGTACAGCTC…
Representative Results
Figure 1. SOD1 WT and A4V stable line generation. (A) Schematic diagram of lentiviral and packaging vectors (Packing and Envelop plasmids) for wild type and mutant SOD1 A4V lentivirus generation. (B) Selected confocal images of three cells (HEK-293, U2OS, and SH-SY5Y) transduced with the lentivirus wild type SOD1 (WT) and mutant SOD1 (A4V). (C) Representative ima…
Declarações
The authors have nothing to disclose.
Materials
| ALLN (C20H37N3O4) | Millipore | 208719 | |
| MG132 (C26H41N3O5) | Sigma-Aldrich | C2211 | |
| Epoxomicin (C28H50N4O7) | Sigma-Aldrich | E3652 | |
| Hoechst 33342 | Invitrogen | H-3570 | |
| Opera | Perkin Elmer | OP-QEHS-01 | |
| Opera EvoShell software | Perkin Elmer | Ver 1.8.1 | |
| Operetta | Perkin Elmer | OPRT1288 | |
| Harmony Imaging software | Perkin Elmer | Ver 3.0.0 | |
| Columbus Image analysis software | Perkin Elmer | Ver 2.3.2 | |
| CyBi Hummingwell liquid handling | CyBio AG | OL 3387 3 0110 |
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Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor. J. Vis. Exp. (Pending Publication), e21172, doi: (2023).