Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations

Polina B Drozdova; Yury A Barbitoff; Mikhail V Belousov; Rostislav K Skitchenko; Tatyana M Rogoza; Jeremy Y Leclercq; Andrey V Kajava; Andrew G Matveenko; Galina A Zhouravleva; Stanislav A Bondarev

doi:10.1080/19336896.2020.1751574

Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations

Prion. 2020 Dec;14(1):118-128. doi: 10.1080/19336896.2020.1751574.

Authors

Polina B Drozdova^{1

2}, Yury A Barbitoff¹, Mikhail V Belousov^{1

3}, Rostislav K Skitchenko⁴, Tatyana M Rogoza^{1

5}, Jeremy Y Leclercq⁶, Andrey V Kajava^{4

6}, Andrew G Matveenko¹, Galina A Zhouravleva^{1

7}, Stanislav A Bondarev^{1

7}

Affiliations

¹ Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg, Russia.
² Institute of Biology, Irkutsk State University, Irkutsk, Russia.
³ Laboratory for Proteomics of Supra-Organismal Systems, All-Russia Research Institute for Agricultural Microbiology, St. Petersburg, Russia.
⁴ International Research Institute of Bioengineering, ITMO University, St. Petersburg, Russia.
⁵ Vavilov Institute of General Genetics Russian Academy of Sciences, St. Petersburg Branch, St. Petersburg, Russia.
⁶ Centre de Recherche En Biologie Cellulaire De Montpellier, UMR 5237 CNRS, Montpellier, France.
⁷ Laboratory of Amyloid Biology, St. Petersburg State University, St. Petersburg, Russia.

Abstract

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

Keywords: Amyloids; Rnq1; SDD-AGE; Shiny application; Sup35.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amyloid / analysis*
Amyloid / ultrastructure
Buffers
Cell Fractionation
Detergents / chemistry*
Electrophoresis, Agar Gel*
Hydrogen-Ion Concentration
Molecular Weight
Protein Aggregates*
Protein Denaturation*
Reproducibility of Results
Saccharomyces cerevisiae / metabolism
Saccharomyces cerevisiae Proteins / chemistry
Saccharomyces cerevisiae Proteins / metabolism
Saccharomyces cerevisiae Proteins / ultrastructure
Spheroplasts / metabolism

Substances

Amyloid
Buffers
Detergents
Protein Aggregates
Saccharomyces cerevisiae Proteins

Grants and funding

This work was supported by the Centre National de la Recherche Scientifique [PRC1524]; Ministry of Science and Higher Education of the Russian Federation [2019-0219]; Russian Science Foundation [18-14-00050]; Russian Science Foundation [19-76-00026]; Saint Petersburg State University [39468250]; Saint Petersburg State University [32693900]; Russian Foundation for Basic Research (RFBR) [19-04-00173]; Russian Foundation for Basic Research (RFBR) [18-34-00537]; Russian Foundation for Basic Research (RFBR) [18-34-00536]; Russian Foundation for Basic Research (RFBR) [17-54-150002].