Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations.
Prion
; 14(1): 118-128, 2020 12.
Article
in En
| MEDLINE
| ID: mdl-32306832
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Protein Denaturation
/
Detergents
/
Electrophoresis, Agar Gel
/
Protein Aggregates
/
Amyloid
Type of study:
Qualitative_research
Language:
En
Journal:
Prion
Journal subject:
BIOQUIMICA
Year:
2020
Document type:
Article
Affiliation country:
Rusia