Strategies to increase the reproducibility of protein fibrillization in plate reader assays.
Anal Biochem
; 400(2): 270-81, 2010 May 15.
Article
en En
| MEDLINE
| ID: mdl-20149780
There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer's and Parkinson's (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in PD and notorious for its erratic fibrillization behavior. Salts and polyanionic polymers do not significantly improve the quality of the assay. However, an orbital agitation mode in the plate reader is a crucial first step toward reproducible alphaSN fibrillization. Higher reproducibility is achieved by the addition of glass beads, as evaluated by the percentage standard deviation of the nucleation and elongation rate constants and the end-stage fluorescence intensity of the fibril-binding dye thioflavin T (ThT). The highest reproducibility is obtained by either seeding the solution with preformed fibrils or by adding submicellar amounts of sodium dodecyl sulfate (SDS), where we obtain percentage standard deviations of 3-4% on the end ThT level. We conclude that there are multiple ways to achieve satisfactory levels of reproducibility, although the different conditions used to induce aggregation may lead to different fibrillization pathways.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Alfa-Sinucleína
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2010
Tipo del documento:
Article
País de afiliación:
Dinamarca
Pais de publicación:
Estados Unidos