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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Published: April 25th, 2021



1Laboratory for Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

Protein aggregations involving neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, and so on1 are known to be toxic and would disturb protein homeostasis (proteostasis) in the cells and organs, that could then lead to aging2. The clearance of protein aggregation is expected as a therapeutic strategy; however, chemicals that prevent protein aggregation formation and degrade protein aggregates (e.g., small molecules or drugs) have not been established yet. Moreover, how protein aggregation exerts toxicity remains elusive. Therefore, to pro....

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1. Materials and reagents

  1. Use pyrogenic free solutions and medium for cell culture (Table 1).
  2. Prepare solutions for the biochemical experiment using ultrapure water and use as DNase/RNase free.
  3. Select an appropriate FBS for the cell culture with a lot check process. Since the selected FBS lot changes regularly, the catalog and lot number for FBS cannot be represented here.
  4. Plasmid DNA
    1. Prepare pmeGFP-N15 for eGFP monomer express.......

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We performed FCS measurement of GFP-TDP25 in cell lysate and SOD1-G85R-GFP in live cells. In both cases, a positive amplitude and smooth ACFs were able to be acquired. We have shown that a portion of GFP-TDP25 expressed in Neuro2a cells was recovered in the soluble fraction under the indicated condition6. In the soluble fraction of the cell lysate, extremely bright fluorescence molecules were detected in the photon count rate record using FCS (Figure 2A, top, arrow). .......

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Regarding system calibration before measurements, the same glasswares as the one used to measure the sample should be used (e.g., the 8-wells cover glass chamber for cell lysate and the 35-mm glass base dish for live cells). Because of the adsorption of Rh6G on the glass, its effective concentration may sometimes decrease. If so, a highly concentrated Rh6G solution such as 1 μM should be used just for the pinhole adjustment. Extremely high photon count rates must be avoided to protect the detector (e.g., more than 1.......

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A.K. was supported by a Japan Society for Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) (#18K06201), by a grant-in-aid from the Nakatani Foundation for Countermeasures against novel coronavirus infections, by a grant from Hokkaido University Office for Developing Future Research Leaders (L-Station), and a grant-in-aid from Hoansha Foundation. M. K. was partially supported by a JSPS Grant-in-Aid for Scientific Research on Innovative Areas "Chemistry for Multimolecular Crowding Biosystems" (#20H04686), and a JSPS Grant-in-Aid for Scientific Research on Innovative Areas "Information physics of living matters" (#20H05522).


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Name Company Catalog Number Comments
0.25% (w/v) Trypsin-1 mmol/L EDTA·4Na Solution with Phenol Red (Trypsin-EDTA) Fujifilm Wako Pure Chemical Corp. 201-16945
100-mm plastic dishes CORNING 430167
35-mm glass base dish IWAKI 3910-035 For live cell measurement
35-mm plastic dishes Thermo Fisher Scientific 150460
Aluminum plate Bio-Bik AB-TC1
C-Apochromat 40x/1.2NA Korr. UV-VIS-IR M27 Carl Zeiss Objective
Cell scraper Sumitomo Bakelite Co., Ltd. MS-93100
Cellulose acetate filter membrane (0.22 mm) Advantech Toyo 25CS020AS
Cover glass chamber 8-wells IWAKI 5232-008 For solution measurement
Dulbecco's Modified Eagle's Medium (DMEM) Sigma-Aldrich D5796 basal medium
Fetal bovine serum (FBS) biosera Lot check required
Lipofectamine 2000 Thermo Fisher Scientific 11668019
LSM510 META + ConfoCor3 Carl Zeiss FCS system
Murine neuroblastoma Neuro2a cells ATCC CCL-131 Cell line
Opti-MEM I Thermo Fisher Scientific 31985070
pCAGGS RIKEN RDB08938 Plasmid DNA for the transfection carrier
Penicillin-Streptomycin Solution (×100 ) Fujifilm Wako Pure Chemical Corp. 168-23191
pmeGFP-C1-TDP25 Plasmid DNA for TDP25 tagged with monomeric eGFP
pmeGFP-N1 Plasmid DNA for eGFP monomer expression
pmeGFP-N1-SOD1-G85R Plasmid DNA for ALS-linked G85R mutant of SOD1 tagged with monomeric eGFP
Protease inhibitor cocktail Sigma-Aldrich P8304

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