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

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Overview

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 CCGCTACTTGTACAGCTCGTCCATGCC-3′and inserted into the pTRIP-delta U3 CMV plasmid using XhoI and BsrGI restriction sites. Avoidance of more than 20 passages and main....

Results

figure-results-58
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.......

Disclosures

No conflicts of interest declared.

Materials

Name	Company	Catalog Number	Comments
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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Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)